MMSC2 - an MMAC1 interacting protein

ABSTRACT

The present invention is directed to the MMSC2 gene, its protein product and the use of the protein to (i) detect mutant MMAC1 proteins, (ii) screen for drugs which can be used for suppressing tumor growth and (iii) identify proteins which interact with the MMAC1 gene or are involved in the tumor suppression pathway of the MMAC1 gene.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is a divisional application of U.S.patent application Ser. No. 09/306,998 filed May 7, 1999. The presentapplication is related to U.S. provisional patent application Ser. No.60/084,740, filed May 8, 1998, incorporated herein by reference, andclaims priority thereto under 35 USC § 119(e).

BACKGROUND OF THE INVENTION

[0002] The present invention is directed to the MMSC2 gene, its proteinproduct and the use of the protein to (i) detect mutant MMAC1 proteins,(ii) screen for drugs which can be used for suppressing tumor growth and(iii) identify proteins which interact with the MMAC1 gene or areinvolved in the tumor suppression pathway of the MMAC1 gene.

[0003] The publications and other materials used herein to illuminatethe background of the invention or provide additional details respectingthe practice, are incorporated by reference, and for convenience arerespectively grouped in the appended List of References.

[0004] A number of genetic alterations are involved in the oncogenesisof glioblastoma multiforme, including inactivation of p53, p16, RB,amplification of the gene encoding epidermal growth factor receptor andseveral other molecular alterations (Louis & Gusella, 1995). However themost common genetic alteration is the deletion of large regions or anentire copy of chromosome 10 (Fults et al., 1990; Rahseed et al., 1992).Recently, the tumor suppressor gene MMAC1 (Steck et al., 1997), alsoknown as PTEN (Li et al., 1997) or TEP1 (Li & Sun, 1997) was mapped to10q23 and shown to be mutated in 17-24% of xenografted and primaryglioblastomas, 14% of breast cancer samples and 25% of kidney carcinomas(Steck et al., 1997). The mutation frequency in established cell linesof these tumor types is somewhat higher. In addition to this predictedinvolvement in sporadic cancer, germ-line MMAC1 mutations have beendetected in two autosomal dominant disorders, Cowden disease (Nelen etal., 1997; Liaw et al., 1997), a syndrome that confers an elevated riskfor tumors of breast, thyroid and skin, and Bannayan-Zonana syndrome(Marsh et al., 1997), a condition characterized by macrocephaly,lipomas, intestinal hamartomatous polyps, vascular malformations andsome skin disorders. Mutations of MMAC1 in primary endometrialcarcinomas (Kong et al., 1997) and in juvenile polyposis coli (Olschwanget al., 1998) have also been seen.

[0005] The predicted protein product of the MMAC1 gene has severalregions of homology with other proteins. The MMAC1 protein has an animoterminal domain with extensive homology to tensin, a protein thatinteracts with actin filaments at focal adhesions, and with auxilin, aprotein involved in synaptic vesicle transport. The MMAC1 protein alsohas a region with extensive homology to protein tyrosine phosphatases(Steck et al., 1997; Li et al., 1997). Mutations of MMAC1 in tumors, itscytoplasmic localization (Li & Sun, 1997) and its intrinsic phosphataseactivity (Li & Sun, 1997; Myers et al., 1997) suggested that itsactivity could be important in some aspect of tumor progression,possibly to counteract the oncogenic effect of a specific proteintyrosine kinase. In addition, MMAC1 is rapidly down-regulated by TGFβ incells sensitive to its cell growth and cell adhesion regulatoryproperties (Li & Sun, 1997).

[0006] Experiments on glioma cell growth have shown that MMAC1 is aprotein phosphatase that exhibits functional and specificgrowth-suppressing activity. In such experiments, the introduction ofHA-tagged MMAC1 into glioma cells containing endogenous mutant allelescaused growth suppression, but was without effect in cells containingwild-type MMAC1 (Furnari et al., 1997). The ectopic expression of MMAC1alleles, which carried mutations found in primary tumors and have beenshown or are expected to inactivate its phosphatase activity, causedlittle growth suppression (Furnari et al., 1997). Although theseactivities of MMAC1 are known, the mechanisms of tumor suppression byMMAC1 and the interaction of the MMAC1 protein with other proteins arenot well understood.

[0007] Many cytosolic signaling proteins and cytoskeletal proteins arecomposed of modular units of small protein-protein interactive domainsthat allow reversible and regulated assembly into larger proteincomplexes. These domains include the Src-homology SH2 and SH3 domains(Schlessinger, 1994; Pawson, 1994), pleckstrin-homology (PH) domains(Lemmon et al., 1996; Shaw, 1996), phosphotyrosine-binding (PTB) domains(Harrison, 1996; van der Greer & Pawson, 1995; Kavanaugh et al., 1995)and postsynaptic density protein, disc-large, zo-1 (PDZ) domains (Woods& Bryant, 1991; Dho et al., 1992; Woods & Bryant, 1993; Kennedy, 1995;Komau et al., 1995). So far, PDZ domains have been found in more than 50proteins (Tsunoda et al., 1997), and many proteins have multiple PDZdomains (Pawson & Scott, 1997). For a review of PDZ domains, as well asthe other protein-protein interactive domains, see Pawson & Scott(1997).

[0008] A distinguishing feature of PDZ domains is their recognition ofshort peptides at the carboxyl terminal end of proteins. For example,one family of PDZ domains selected peptides with the consensus motifGlu-(Ser/Thr)-Xaa-(Val/Ile) (SEQ ID NO: 1) at the carboxy terminus,whereas a second family of PDZ domains selected peptides withhydrophobic or aromatic side chains at the carboxy terminal threeresidues (Songyang et al., 1997). The presence of multiple PDZ domainsin proteins may have at least two important consequences. An individualPDZ-containing protein could bind several subunits of a particularchannel thereby inducing channel aggregations. Furthermore, theindividual domains of a protein can have distinct binding specificitiesthereby inducing the formation of clusters that contain heterogeneousgroups of proteins.

[0009] One example of this latter consequence of multiple PDZ domains isthe InaD protein which contains five PDZ domains and acts as ascaffolding protein to organize the light-activated signaling events inDrosophila (Shieh & Zhu, 1996; Tsunoda et al., 1997). InaD associatesthrough distinct PDZ domains with a calcium channel(TRP), phospholipaseC-β (the target of rhodopsin-activated heterotrimeric guaninenucleotide-binding protein (Gqα) and protein kinase C.

[0010] Two further properties of PDZ domains or proteins which containthem may expand their potential activities. First, some PDZ domains maybind internal peptide sequences and, indeed, have a propensity toundergo homotypic or heterotypic interactions with other PDZ domains(Brenman et al., 1996). Second, proteins with PDZ domains frequentlycontain other interaction modules, including SH3 and LIM domains, andcatalytic elements such a tyrosine phosphatase or nitric oxide synthasedomains. PDZ domains may therefore both coordinate the localization andclustering of receptors and channels, and provide a bridge to thecytoskeleton or intracellular signaling pathways.

[0011] It is desired to determine the mechanisms of tumor suppressionfor MMAC1 and to identify proteins which interact with the MMAC1protein. Such proteins can be used to assay for mutated MMAC1 proteinsand/or screen potential drugs for suppressing tumor growth and/oridentify additional proteins which interact with MMAC1.

SUMMARY OF THE INVENTION

[0012] The present invention is directed to the MMSC2 gene, its proteinproduct and the use of the protein to (i) detect mutant MMAC1 proteins,(ii) screen for drugs which can be used for suppressing tumor growth and(iii) identify proteins which interact with the MMAC1 gene or areinvolved in the tumor suppression pathway of the MMAC1 gene.

[0013] Using yeast two-hybrid screening, it has been found MMAC1 bindsto a protein herein named MMSC2. The nucleotide sequence is set forth asSEQ ID NO: 2, and the amino acid sequence is set forth as SEQ ID NO: 3.It has been found that MMSC2 has 11 PDZ domains and that one or more ofthese domains interacts specifically with the three carboxyl terminalamino acids of MMAC1. Specifically, it has been found that PDZ domainnumbers 7,10 and 13 interact with MMAC1, with 7 appearing stronger.Since MMSC2 contains 11 PDZ domains and interacts with MMAC1, a knowntumor suppressor having a region of homology with protein tyrosinephosphatases, MMSC2 acts as a scaffolding protein in a commonbiochemical pathway with MMAC1. These characteristics indicate that theinteraction between MMAC1 and MMSC2 is required for the tumor suppressoractivity of MMAC1.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

[0014]FIG. 1 shows a diagram of MMSC2 indicating the position of ORF andthe positions of the 13 PDZ domains.

[0015]FIG. 2 shows a diagram of the key clones used to assemble the fulllength MMSC2 seqeunce, the probes used to identify those clones, and therelative position of the partially sequenced mouse ortholog9BP-1(GenBank Accession # AF000168).

SUMMARY OF SEQUENCE LISTING

[0016] SEQ ID NO: 1 is a consensus motif to which one family of PDZdomains interacts. SEQ ID NO: 2 is the nucleotide sequence for the MMSC2gene. SEQ ID NO: 3 is the amino acid sequence for the MMSC2 protein. SEQID NO: 4 is the 15 C-terminal amino acids of MMAC1. SEQ ID NO: 5 isprimer 9BP-1 F1. SEQ ID NO: 6 is primer 9BP-1 R4. SEQ ID NO: 7 is primer9BP-1 #1. SEQ ID NO: 8 is primer 9BP-1 #2. SEQ ID NO: 9 is primer 9BP-1#5. SEQ ID NO: 10 is primer 9BP-1 #7. SEQ ID NO: 11 is the SH3 bindingpeptide. SEQ ID NO: 12 is the MMAC1 binding peptide. SEQ ID NOs: 13-72are primers for PCR amplification of the MMSC2 gene.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention is directed to the MMSC2 gene, its proteinproduct and the use of the protein to (i) detect mutant MMAC1 proteins,(ii) screen for drugs which can be used for suppressing tumor growth and(iii) identify proteins which interact with the MMAC1 gene or areinvolved in the tumor suppression pathway of the MMAC1 gene.

[0018] Using yeast two-hybrid screening, it has been found MMAC1 bindsto a protein herein named MMSC2. The nucleotide sequence is set forth asSEQ ID NO: 2, and the amino acid sequence is set forth as SEQ ID NO: 3.It has been found MMSC2 has 11 PDZ domains and that one or more of thesedomains interacts specifically with the three carboxyl terminal aminoacids of MMAC1. Specifically, it has been found that PDZ domain numbers7, 10 and 13 interact with MMAC1 with 7 appearing stronger. Since MMSC2contains 11 PDZ domains and interacts with MMAC1, a known tumorsuppressor having a region of homology with protein tyrosinephosphatases, MMSC2 acts as a scaffolding protein in a commonbiochemical pathway with MMAC1. These characteristics indicate that theinteraction between MMAC1 and MMSC2 is required for the tumor suppressoractivity of MMAC1.

[0019] The evidence presented herein shows that the function of MMSC2 isto make a scaffold that binds to MMAC1, the phosphatase substrate(s),and the (probably oncogene) tyrosine kinase(s). Thus, a valuable drugwill be one that can prevent binding of either the substrate(s) or thetyrosine kinases(s) to MMSC2.

[0020] The yeast two-hybrid screening assay described herein identifiedfive clones encoding bona fide MMAC1-interacting proteins. These cloneswere named PDZBN2B, PDZBN3A, PDZBN5B, PDZBN18D, and pdzk4. Comparison ofthe sequences of these clones suggested that they were all partial cDNAsderived from the same novel gene. A search of GenBank with thesesequences revealed strong sequence similarity with a partial mouse cDNAsequence called 9ORF binding protein 1 (9BP-1)(GenBank Accession #AF000168).

[0021] Several rounds of cDNA library screening were required toidentify cDNA clones that could be assembled into the full length MMSC2sequence. In the first round, a 509 base pair(bp) probe was developedfrom the 5′ end of clone PDZBN2B using the primers 9BP-1 F1and 9BP-1 R4.This probe was used to screen a human placental cDNA library and a humanprostate cDNA library. Two of the informative clones obtained were p118a(placental) and pr63(prostate). A search of GenBank with this additionalsequence yielded an additional human EST (GenBank Accesion # C75629).For the second round of cDNA library screening, a 202 bp probe wasdeveloped from the 5′ end of this EST using primers 9BP-1 #1 and 913P-1#2. This probe was used to screen a human prostate cDNA library; two ofthe informative clones obtained were clone #10 and clone #3. For thethird round of cDNA library screening, a 172 bp probe was developed fromthe 5′ end of clone #3 using primers 9BP-1 #5 and 9BP-1 #7 and used toscreen a human prostate cDNA library. One of the resulting clones, clone# 6, yielded the start codon and part of the 5′ UTR, including in-frameupstream stop codons. The nucleotide sequence for MMSC2 is set forth inSEQ ID NO: 2 with the amino acid sequence of the encoded protein setforth in SEQ ID NO: 3.

[0022] As previously noted, SEQ ID NO: 2 sets forth the nucleotidesequence for MMSC2. However, it has been found that the mRNA for MMSC2is subject to alternate splicing. On the basis of the sequence forMMSC2, genomic clones have been isolated and are being analyzed todetermine splice junctions and alternate splicing for the mRNA. Inaddition, the PDZ domains of MMSC2 are analyzed in the yeast two-hybridassay to identify other proteins which interact with MMSC2 andconsequently are involved in the MMAC1 tumor suppressor pathway.

[0023] Since MMSC2 is an MMAC1 interacting protein that is involved intumor suppression activity in the MMAC1 pathway, mutations in the MMSC2gene which affect the interaction of MMSC2 with MMAC1 or affect theinteraction of other proteins with MMAC1 as a result of the scaffoldingeffect of MMSC2 will interfere with the MMAC1 tumor suppressor pathwayand lead to tumorigenesis. Thus, an additional aspect of the presentinvention is the screening of MMSC2 for such mutations usingconventional techniques. Such methods may further comprise the step ofamplifying a portion of the MMSC2 gene, and may further include a stepof providing a set of polynucleotides which are primers foramplification of said portion of the MMSC2 gene. The method is usefulfor identifying mutations for use in either diagnosis of cancer orprognosis of cancer. Since such variants can now be detected earlier,i.e., before symptoms appear, and more definitively, better treatmentoptions will be available in those individuals identified as havingharmful mutations in MMSC2.

[0024] The present invention is directed to the determination that theMMSC2 binds to the C-terminal region of MMAC1 and is involved in acommon pathway with MMAC1 which is a known tumor suppressor. Since manyof the mutations in MMAC1 are frameshift or nonsense mutations whichconsequently alter the C-terminus of MMAC1, MMSC2 can be used to assayfor normal or mutated MMAC1 proteins using conventional techniques.

[0025] Finally, the present invention is directed to a method forscreening drug candidates to identify drugs useful for treating orpreventing cancer. Drug screening is performed by expressing mutantMMSC2 and assaying the effect of a drug candidate on the binding ofMMSC2 with MMAC1. Similarly, one can test the effect of a drug candidateon the binding of wild-type MMSC2 with a mutant MMAC1. Such assays canbe performed in vitro or in vivo, such as in oocytes, mammalian cells ortransgenic animals. Other assays may test the ability of a drug, whereinthe drug may be, e.g., a peptide, to replace the activity of MMSC2 suchthat the drug plus MMAC1 will work in concert similar to the normalwild-type interactions of MMSC2 and MMAC1. Again, similar assays may beperformed to screen for drugs which replace a mutant MMAC1 and will bindto wild-type MMSC2 to replace the MMAC1 function which is lacking as aresult of a mutated MMAC1.

[0026] According to the diagnostic and prognostic method of the presentinvention, alteration of the wild-type MMSC2 gene is detected. Inaddition, the method can be performed by detecting the wild- type MMSC2gene and confirming the lack of a cause of cancer as a result of thislocus. “Alteration of a wild-type gene” encompasses all forms ofmutations including deletions, insertions and point mutations in thecoding and noncoding regions. Deletions may be of the entire gene or ofonly a portion of the gene. Point mutations may result in stop codons,frameshift mutations or amino acid substitutions. Somatic mutations arethose which occur only in certain tissues and are not inherited in thegermline. Germline mutations can be found in any of a body's tissues andare inherited. Point mutational events may occur in regulatory regions,such as in the promoter of the gene, leading to loss or diminution ofexpression of the mRNA. Point mutations may also abolish proper RNAprocessing, leading to loss of expression of the MMSC2 gene product, orto a decrease in mRNA stability or translation efficiency.

[0027] Useful diagnostic techniques include, but are not limited tofluorescent in situ hybridization (FISH), direct DNA sequencing, PFGEanalysis, Southern blot analysis, single stranded conformation analysis(SSCA), RNase protection assay, allele-specific oligonucleotide (ASO),dot blot analysis, hybridization using nucleic acid modified with goldnanoparticles and PCR-SSCP, as discussed in detail further below. Alsouseful is the recently developed technique of DNA microchip technology.

[0028] The presence of cancer due to a germline mutation at this locusmay be ascertained by testing any tissue of a human for mutations of theMMSC2 gene. For example, a person who has inherited a germline MMSC2mutation, especially one which alters the interaction of MMSC2 withMMAC1, would be prone to develop cancer. This can be determined bytesting DNA from any tissue of the person's body. Most simply, blood canbe drawn and DNA extracted from the cells of the blood. In addition,prenatal diagnosis can be accomplished by testing fetal cells, placentalcells or amniotic cells for mutations of the MMSC2 gene. Alteration of awild-type MMSC2 allele, whether, for example, by point mutation ordeletion, can be detected by any of the means discussed herein.

[0029] There are several methods that can be used to detect DNA sequencevariation. Direct DNA sequencing, either manual sequencing or automatedfluorescent sequencing can detect sequence variation. Another approachis the single-stranded conformation polymorphism assay (SSCP) (Orita etal., 1989). This method does not detect all sequence changes, especiallyif the DNA fragment size is greater than 200 bp, but can be optimized todetect most DNA sequence variation. The reduced detection sensitivity isa disadvantage, but the increased throughput possible with SSCP makes itan attractive, viable alternative to direct sequencing for mutationdetection on a research basis. The fragments which have shifted mobilityon SSCP gels are then sequenced to determine the exact nature of the DNAsequence variation. Other approaches based on the detection ofmismatches between the two complementary DNA strands include clampeddenaturing gel electrophoresis (CDGE) (Sheffield et al., 1991),heteroduplex analysis (HA) (White et al., 1992) and chemical mismatchcleavage (CMC) (Grompe et al., 1989). None of the methods describedabove will detect large deletions, duplications or insertions, nor willthey detect a regulatory mutation which affects transcription ortranslation of the protein. Other methods which might detect theseclasses of mutations such as a protein truncation assay or theasymmetric assay, detect only specific types of mutations and would notdetect missense mutations. A review of currently available methods ofdetecting DNA sequence variation can be found in a recent review byGrompe (1993). Once a mutation is known, an allele specific detectionapproach such as allele specific oligonucleotide (ASO) hybridization canbe utilized to rapidly screen large numbers of other samples for thatsame mutation. Such a technique can utilize probes which are labeledwith gold nanoparticles to yield a visual color result (Elghanian etal., 1997).

[0030] A rapid preliminary analysis to detect polymorphisms in DNAsequences can be performed by looking at a series of Southern blots ofDNA cut with one or more restriction enzymes, preferably with a largenumber of restriction enzymes. Each blot contains a series of normalindividuals and a series of cancer cases. Southern blots displayinghybridizing fragments differing in length from control DNA when probedwith sequences near or including the MMSC2 locus indicate a possiblemutation. If restriction enzymes which produce very large restrictionfragments are used, then pulsed field gel electrophoresis (PFGE) isemployed.

[0031] Detection of point mutations may be accomplished amplification,e.g., PCR, from genomic or cDNA and sequencing the amplified nucleicacid or by molecular cloning of the MMSC2 allele and sequencing theallele using techniques well known in the art.

[0032] There are six well known methods for a more complete, yet stillindirect, test for confirming the presence of a susceptibilityallele: 1) single stranded conformation analysis (SSCP) (Orita et al.,1989); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell etal., 1990; Sheffield et al., 1989); 3) RNase protection assays(Finkelstein et al., 1990; Kinszler et al., 1991); 4) allele-specificoligonucleotides (ASOs) (Conner et al., 1983); 5) the use of proteinswhich recognize nucleotide mismatches, such as the E. coli mutS protein(Modrich, 1991); and 6) allele-specific PCR (Rano and Kidd, 1989). Forallele-specific PCR, primers are used which hybridize at their 3′ endsto a particular MMSC2 mutation. If the particular mutation is notpresent, an amplification product is not observed. AmplificationRefractory Mutation System (ARMS) can also be used, as disclosed inEuropean Patent Application Publication No. 0332435 and in Newton etal., 1989. Insertions and deletions of genes can also be detected bycloning, sequencing and amplification. In addition, restriction fragmentlength polymorphism (RFLP) probes for the gene or surrounding markergenes can be used to score alteration of an allele or an insertion in apolymorphic fragment. Such a method is particularly useful for screeningrelatives of an affected individual for the presence of the mutationfound in that individual. Other techniques for detecting insertions anddeletions as known in the art can be used.

[0033] In the first three methods (SSCP, DGGE and RNase protectionassay), a new electrophoretic band appears. SSCP detects a band whichmigrates differentially because the sequence change causes a differencein single-strand, intramolecular base pairing. RNase protection involvescleavage of the mutant polynucleotide into two or more smallerfragments. DGGE detects differences in migration rates of mutantsequences compared to wild-type sequences, using a denaturing gradientgel. In an allele-specific oligonucleotide assay, an oligonucleotide isdesigned which detects a specific sequence, and the assay is performedby detecting the presence or absence of a hybridization signal. In themutS assay, the protein binds only to sequences that contain anucleotide mismatch in a heteroduplex between mutant and wild-typesequences.

[0034] Mismatches, according to the present invention, are hybridizednucleic acid duplexes in which the two strands are not 100%complementary. Lack of total homology may be due to deletions,insertions, inversions or substitutions. Mismatch detection can be usedto detect point mutations in the gene or in its mRNA product. Whilethese techniques are less sensitive than sequencing, they are simpler toperform on a large number of samples. An example of a mismatch cleavagetechnique is the RNase protection method. In the practice of the presentinvention, the method involves the use of a labeled riboprobe which iscomplementary to the human wild-type MMSC2 gene coding sequence. Theriboprobe and either mRNA or DNA isolated from the person are annealed(hybridized) together and subsequently digested with the enzyme RNase Awhich is able to detect some mismatches in a duplex RNA structure. If amismatch is detected by RNase A, it cleaves at the site of the mismatch.Thus, when the annealed RNA preparation is separated on anelectrophoretic gel matrix, if a mismatch has been detected and cleavedby RNase A, an RNA product will be seen which is smaller than the fulllength duplex RNA for the riboprobe and the mRNA or DNA. The riboprobeneed not be the fall length of the mRNA or gene but can be a segment ofeither. If the riboprobe comprises only a segment of the mRNA or gene,it will be desirable to use a number of these probes to screen the wholemRNA sequence for mismatches.

[0035] In similar fashion, DNA probes can be used to detect mismatches,through enzymatic or chemical cleavage. See, e.g., Cotton et al., 1988;Shenk et al., 1975; Novack et al., 1986. Alternatively, mismatches canbe detected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, 1988. Witheither riboprobes or DNA probes, the cellular mRNA or DNA which mightcontain a mutation can be amplified using PCR (see below) beforehybridization. Changes in DNA of the MMSC2 gene can also be detectedusing Southern hybridization, especially if the changes are grossrearrangements, such as deletions and insertions.

[0036] DNA sequences of the MMSC2 gene which have been amplified by useof PCR may also be screened using allele-specific probes. These probesare nucleic acid oligomers, each of which contains a region of the genesequence harboring a known mutation. For example, one oligomer may beabout 30 nucleotides in length, corresponding to a portion of the genesequence. By use of a battery of such allele-specific probes, PCRamplification products can be screened to identify the presence of apreviously identified mutation in the gene. Hybridization ofallele-specific probes with amplified MMSC2 sequences can be performed,for example, on a nylon filter. Hybridization to a particular probeunder high stringency hybridization conditions indicates the presence ofthe same mutation in the tissue as in the allele-specific probe.

[0037] The newly developed technique of nucleic acid analysis viamicrochip technology is also applicable to the present invention. Inthis technique, literally thousands of distinct oligonucleotide probesare built up in an array on a silicon chip. Nucleic acid to be analyzedis fluorescently labeled and hybridized to the probes on the chip. It isalso possible to study nucleic acid-protein interactions using thesenucleic acid microchips. Using this technique one can determine thepresence of mutations or even sequence the nucleic acid being analyzedor one can measure expression levels of a gene of interest. The methodis one of parallel processing of many, even thousands, of probes at onceand can tremendously increase the rate of analysis. Several papers havebeen published which use this technique. Some of these are Hacia et al.,1996; Shoemaker et al., 1996; Chee et al., 1996; Lockhart et al., 1996;DeRisi et al., 1996; Lipshutz et al., 1995. This method has already beenused to screen people for mutations in the breast cancer gene BRCA1(Hacia et al., 1996). This new technology has been reviewed in a newsarticle in Chemical and Engineering News (Borman, 1996) and been thesubject of an editorial (Nature Genetics, 1996). Also see Fodor (1997).

[0038] The most definitive test for mutations in a candidate locus is todirectly compare genomic MMSC2 sequences from patients with those from acontrol population. Alternatively, one could sequence messenger RNAafter amplification, e.g., by PCR, thereby eliminating the necessity ofdetermining the exon structure of the candidate gene.

[0039] Mutations from patients falling outside the coding region ofMMSC2 can be detected by examining the non-coding regions, such asintrons and regulatory sequences near or within the genes. An earlyindication that mutations in noncoding regions are important may comefrom Northern blot experiments that reveal messenger RNA molecules ofabnormal size or abundance in patients as compared to controlindividuals.

[0040] Alteration of MMSC2 mRNA expression can be detected by anytechniques known in the art. These include Northern blot analysis, PCRamplification and RNase protection. Diminished mRNA expression indicatesan alteration of the wild-type gene. Alteration of wild-type genes canalso be detected by screening for alteration of wild-type MMSC2 protein.For example, monoclonal antibodies immunoreactive with MMSC2 can be usedto screen a tissue. Lack of cognate antigen would indicate a mutation.Antibodies specific for products of mutant alleles could also be used todetect mutant gene product. Such immunological assays can be done in anyconvenient formats known in the art. These include Western blots,immunohistochemical assays and ELISA assays. Any means for detecting analtered MMSC2 protein can be used to detect alteration of the wild-typeMMSC2 gene. Functional assays, such as protein binding determinations,can be used. In addition, assays can be used which detect MMSC2biochemical function. Finding a mutant MMSC2 gene product indicatesalteration of a wild-type MMSC2 gene. One such binding assay is thebinding of MMSC2 with wild-type MMAC1. Conversely, wild-type MMSC2 orthe PDZ domain interacting with MMAC1 can be used in a protein bindingassay or biochemical function assay to detect normal or mutant MMAC1proteins, where the mutant proteins are proteins lacking a wild-typeC-terminus.

[0041] A mutant MMSC2 gene or gene product or a mutant MMAC1 can also bedetected in other human body samples, such as serum, stool, urine andsputum. The same techniques discussed above for detection of mutantgenes or gene products in tissues can be applied to other body samples.By screening such body samples, a simple early diagnosis can be achievedfor cancer resulting from a mutation in the MMSC2 gene.

[0042] The primer pairs of the present invention are useful fordetermination of the nucleotide sequence of a particular MMSC2 alleleusing PCR. The pairs of single-stranded DNA primers for MMSC2 can beannealed to sequences within or surrounding the MMSC2 gene in order toprime amplifying DNA synthesis of the gene itself. A complete set ofthese primers allows synthesis of all of the nucleotides of the genecoding sequences, i.e., the exons. The set of primers preferably allowssynthesis of both intron and exon sequences. Allele-specific primers canalso be used. Such primers anneal only to particular MMSC2 mutantalleles, and thus will only amplify a product in the presence of themutant allele as a template.

[0043] In order to facilitate subsequent cloning of amplified sequences,primers may have restriction enzyme site sequences appended to their 5′ends. Alternatively, primers can also be prepared with 5′ phosphorylgroups which will allow for blunt end coloning of amplied sequences.Thus, all nucleotides of the primers are derived from MMSC2 sequence orsequences adjacent to MMSC2, except for the few nucleotides necessary toform a restriction enzyme site. Such enzymes and sites are well known inthe art. The primers themselves can be synthesized using techniqueswhich are well known in the art. Generally, the primers can be madeusing oligonucleotide synthesizing machines which are commerciallyavailable. Given the sequence of MMSC2, design of particular primers iswell within the skill of the art.

[0044] The nucleic acid probes provided by the present invention areuseful for a number of purposes. They can be used in Southernhybridization to genomic DNA and in the RNase protection method fordetecting point mutations already discussed above. The probes can beused to detect PCR amplification products. They may also be used todetect mismatches with the MMSC2 gene or mRNA using other techniques.

[0045] Mutations which interfere with the function of the MMSC2 geneproduct are involved in the pathogenesis of cancer. Thus, the presenceof an altered (or a mutant) MMSC2 gene which produces a protein having aloss of function, or altered function, directly increases the risk ofcancer. In order to detect a MMSC2 gene mutation, a biological sample isprepared and analyzed for a difference between the sequence of theallele being analyzed and the sequence of the wild-type allele. MutantMMSC2 alleles can be initially identified by any of the techniquesdescribed above. The mutant alleles are then sequenced to identify thespecific mutation of the particular mutant allele. Alternatively, mutantalleles can be initially identified by identifying mutant (altered)proteins, using conventional techniques. The mutant alleles are thensequenced to identify the specific mutation for each allele. Themutations, especially those which lead to an altered function of theprotein, are then used for the diagnostic and prognostic methods of thepresent invention.

[0046] Definitions

[0047] The present invention employs the following definitions.

[0048] “Amplification of Polynucleotides” utilizes methods such as thepolymerase chain reaction (PCR), ligation amplification (or ligase chainreaction, LCR) and amplification methods based on the use of Q-betareplicase. Also useful are strand displacement amplification (SDA),thermophilic SDA, nucleic acid sequence based amplification (3SR orNASBA) and repair chain reaction (RCR). These methods are well known andwidely practiced in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and4,683,202 and Innis et al., 1990 (for PCR); Wu et al., 1989a and EP320,308A (for LCR); U.S. Pat. Nos. 5,270,184 and 5,455,166 and Walker etal., 1992 (for SDA); Spargo et al., 1996 (for thermophilic SDA) and U.S.Pat. No. 5,409,818, Fahy et al., 1991 and Compton, 1991 for 3SR andNASBA. Reagents and hardware for conducting PCR are commerciallyavailable. Primers useful to amplify sequences from the MMSC2 region arepreferably complementary to, and hybridize specifically to sequences inthe MMSC2 region or in regions that flank a target region therein. MMSC2sequences generated by amplification may be sequenced directly.Alternatively, but less desirably, the amplified sequence(s) may becloned prior to sequence analysis. A method for the direct cloning andsequence analysis of enzymatically amplified genomic segments has beendescribed by Scharf, 1986.

[0049] “Analyte polynucleotide” and “analyte strand” refer to a single-or double-stranded polynucleotide which is suspected of containing atarget sequence, and which may be present in a variety of types ofsamples, including biological samples.

[0050] “Antibodies.” The present invention also provides polyclonaland/or monoclonal antibodies and fragments thereof, and immunologicbinding equivalents thereof, which are capable of specifically bindingto the MMSC2 polypeptide and fragments thereof or to polynucleotidesequences from the MMSC2 region. The term “antibody” is used both torefer to a homogeneous molecular entity, or a mixture such as a serumproduct made up of a plurality of different molecular entities.Polypeptides may be prepared synthetically in a peptide synthesizer andcoupled to a carrier molecule (e.g., keyhole limpet hemocyanin) andinjected over several months into rabbits. Rabbit sera is tested forimmunoreactivity to the MMSC2 polypeptide or fragment. Monoclonalantibodies may be made by injecting mice with the protein polypeptides,fusion proteins or fragments thereof. Monoclonal antibodies will bescreened by ELISA and tested for specific immunoreactivity with MMSC2polypeptide or fragments thereof. See, Harlow and Lane, 1988. Theseantibodies will be useful in assays as well as pharmaceuticals.

[0051] Once a sufficient quantity of desired polypeptide has beenobtained, it may be used for various purposes. A typical use is theproduction of antibodies specific for binding. These antibodies may beeither polyclonal or monoclonal, and may be produced by in vitro or invivo techniques well known in the art. For production of polyclonalantibodies, an appropriate target immune system, typically mouse orrabbit, is selected. Substantially purified antigen is presented to theimmune system in a fashion determined by methods appropriate for theanimal and by other parameters well known to immunologists. Typicalsites for injection are in footpads, intramuscularly, intraperitoneally,or intradermally. Of course, other species may be substituted for mouseor rabbit. Polyclonal antibodies are then purified using techniquesknown in the art, adjusted for the desired specificity.

[0052] An immunological response is usually assayed with an immunoassay.Normally, such immunoassays involve some purification of a source ofantigen, for example, that produced by the same cells and in the samefashion as the antigen. A variety of immunoassay methods are well knownin the art. See, e.g., Harlow and Lane, 1988, or Goding, 1986.

[0053] Monoclonal antibodies with affinities of 10⁻⁸ M⁻¹ or preferably10⁻⁹ to 10⁻¹⁰ M⁻¹ or stronger will typically be made by standardprocedures as described, e.g., in Harlow and Lane, 1988 or Goding, 1986.Briefly, appropriate animals will be selected and the desiredimmunization protocol followed. After the appropriate period of time,the spleens of such animals are excised and individual spleen cellsfused, typically, to immortalized myeloma cells under appropriateselection conditions. Thereafter, the cells are clonally separated andthe supernatants of each clone tested for their production of anappropriate antibody specific for the desired region of the antigen.

[0054] Other suitable techniques involve in vitro exposure oflymphocytes to the antigenic polypeptides, or alternatively, toselection of libraries of antibodies in phage or similar vectors. SeeHuse et al., 1989. The polypeptides and antibodies of the presentinvention may be used with or without modification. Frequently,polypeptides and antibodies will be labeled by joining, eithercovalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Suitable labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent agents, chemiluminescent agents,magnetic particles and the like. Patents teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241. Also, recombinant immunoglobulinsmay be produced (see U.S. Pat. No. 4,816,567).

[0055] “Binding partner” refers to a molecule capable of binding aligand molecule with high specificity, as for example, an antigen and anantigen-specific antibody or an enzyme and its inhibitor. In general,the specific binding partners must bind with sufficient affinity toimmobilize the analyte copy/complementary strand duplex (in the case ofpolynucleotide hybridization) under the isolation conditions. Specificbinding partners are known in the art and include, for example, biotinand avidin or streptavidin, IgG and protein A, the numerous, knownreceptor-ligand couples, and complementary polynucleotide strands. Inthe case of complementary polynucleotide binding partners, the partnersare normally at least about 15 bases in length, and may be at least 40bases in length. It is well recognized by those of skill in the art thatlengths shorter than 15 (e.g., 8 bases), between 15 and 40, and greaterthan 40 bases may also be used. The polynucleotides may be composed ofDNA, RNA, or synthetic nucleotide analogs. In addition, as disclosedherein, MMAC1 and PDZ binding peptides, as well as several otherproteins, bind to or interact with MMSC2. Each of these proteins arealso considered binding partners herein. Further binding partners can beidentifed using, e.g., the two-hybrid yeast screening assay as describedherein.

[0056] A “biological sample” refers to a sample of tissue or fluidsuspected of containing an analyte polynucleotide or polypeptide from anindividual including, but not limited to, e.g., plasma, serum, spinalfluid, lymph fluid, the external sections of the skin, respiratory,intestinal, and genitourinary tracts, tears, saliva, blood cells,tumors, organs, tissue and samples of in vitro cell cultureconstituents.

[0057] “Encode”. A polynucleotide is said to “encode” a polypeptide if,in its native state or when manipulated by methods well known to thoseskilled in the art, it can be transcribed and/or translated to producethe mRNA for and/or the polypeptide or a fragment thereof. Theanti-sense strand is the complement of such a nucleic acid, and theencoding sequence can be deduced therefrom.

[0058] “Isolated” or “substantially pure”. An “isolated” or“substantially pure” nucleic acid (e.g., an RNA, DNA or a mixed polymer)is one which is substantially separated from other cellular componentswhich naturally accompany a native human sequence or protein, e.g.,ribosomes, polymerases, many other human genome sequences and proteins.The term embraces a nucleic acid sequence or protein which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems.

[0059] “MMSC2 Allele” refers to normal alleles of the MMSC2 locus thatinteract with MMAC1 as well as alleles of MMSC2 carrying variations thataffect the interaction with MMAC1 and that cause cancer.

[0060] “MMSC2 Locus”, “MMSC2 Gene”, “MMSC2 Nucleic Acids” or “MMSC2Polynucleotide” each refer to polynucleotides, all of which are in theMMSC2 region, that are likely to be expressed in normal tissue, certainalleles of which adversely affect the interaction with MMAC1 and resultin cancer. The MMSC2 locus is intended to include coding sequences,intervening sequences and regulatory elements controlling transcriptionand/or translation. The MMSC2 locus is intended to include all allelicvariations of the DNA sequence.

[0061] These terms, when applied to a nucleic acid, refer to a nucleicacid which encodes a human MMSC2 polypeptide, fragment, homolog orvariant, including, e.g., protein fusions or deletions. The nucleicacids of the present invention will possess a sequence which is eitherderived from, or substantially similar to a natural MMSC2-encoding geneor one having substantial homology with a natural MMSC2-encoding gene ora portion thereof.

[0062] The MMSC2 gene or nucleic acid includes normal alleles of theMMSC2 gene, both silent alleles having no effect on the amino acidsequence of the MMSC2 polypeptide and alleles leading to amino acidsequence variants of the MMSC2 polypeptide that do not substantiallyaffect its function. These terms also include alleles having one or moremutations which adversely affect the function of the MMSC2 polypeptide.A mutation may be a change in the MMSC2 nucleic acid sequence whichproduces a deleterious change in the amino acid sequence of the MMSC2polypeptide, resulting in partial or complete loss of MMSC2 function, ormay be a change in the nucleic acid sequence which results in the lossof effective MMSC2 expression or the production of aberrant forms of theMMSC2 polypeptide.

[0063] The MMSC2 nucleic acid may be that shown in SEQ ID NO: 2, or itmay be an allele as described above, or a variant or derivativediffering from that shown by a change which is one or more of addition,insertion, deletion and substitution of one or more nucleotides of thesequence shown. Changes to the nucleotide sequence may result in anamino acid change at the protein level, or not, as determined by thegenetic code.

[0064] Thus, nucleic acid according to the present invention may includea sequence different from the sequence shown in SEQ ID NO: 2 yet encodea polypeptide with the same amino acid sequence as shown in SEQ ID NO:3. That is, nucleic acids of the present invention include sequenceswhich are degenerate as a result of the genetic code. On the other hand,the encoded polypeptide may comprise an amino acid sequence whichdiffers by one or more amino acid residues from the amino acid sequenceshown in SEQ ID NO: 3. Nucleic acid encoding a polypeptide which is anamino acid sequence variant, derivative or allele of the amino acidsequence shown in SEQ ID NO: 3 is also provided by the presentinvention.

[0065] The MMSC2 gene also refers to (a) any DNA sequence that (i)hybridizes to the complement of the DNA sequences that encode the aminoacid sequence set forth in SEQ ID NO: 3 under highly stringentconditions (Ausubel et al.) (ii) and encodes a gene product functionallyequivalent to MMSC2, or (b) any DNA sequence that (i) hybridizes to thecomplement of the DNA sequences that encode the amino acid sequence setforth in SEQ ID NO: 3 under less stringent conditions, such asmoderately stringent conditions (Ausubel et al.), and (ii) encodes agene product functionally equivalent to MMSC2. The invention alsoincludes nucleic acid molecules that are the complements of thesequences described herein.

[0066] The polynucleotide compositions of this invention include RNA,cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense andantisense strands, and may be chemically or biochemically modified ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those skilled in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

[0067] The present invention provides recombinant nucleic acidscomprising all or part of the MMSC2 region. The recombinant constructmay be capable of replicating autonomously in a host cell.Alternatively, the recombinant construct may become integrated into thechromosomal DNA of the host cell. Such a recombinant polynucleotidecomprises a polynucleotide of genomic, cDNA, RNA, semi-synthetic, orsynthetic origin which, by virtue of its origin or manipulation, 1) isnot associated with all or a portion of a polynucleotide with which itis associated in nature; 2) is linked to a polynucleotide other thanthat to which it is linked in nature; or 3) does not occur in nature.Where nucleic acid according to the invention includes RNA, reference tothe sequence shown should be construed as reference to the RNAequivalent, with U substituted for T.

[0068] Therefore, recombinant nucleic acids comprising sequencesotherwise not naturally occurring are provided by this invention.Although the wild-type sequence may be employed, it will often bealtered, e.g., by deletion, substitution or insertion. cDNA or genomiclibraries of various types may be screened as natural sources of thenucleic acids of the present invention, or such nucleic acids may beprovided by amplification of sequences resident in genomic DNA or othernatural sources, e.g., by PCR. The choice of cDNA libraries normallycorresponds to a tissue source which is abundant in mRNA for the desiredproteins. Phage libraries are normally preferred, but other types oflibraries may be used. Clones of a library are spread onto plates,transferred to a substrate for screening, denatured and probed for thepresence of desired sequences.

[0069] The DNA sequences used in this invention will usually comprise atleast about five codons (15 nucleotides), more usually at least about7-15 codons, and most preferably, at least about 35 codons. One or moreintrons may also be present. This number of nucleotides is usually aboutthe minimal length required for a successful probe that would hybridizespecifically with a MMSC2-encoding sequence. In this context, oligomersof as low as 8 nucleotides, more generally 8-17 nucleotides, can be usedfor probes, especially in connection with chip technology.

[0070] Techniques for nucleic acid manipulation are described generally,for example, in Sambrook et al., 1989 or Ausubel et al., 1992. Reagentsuseful in applying such techniques, such as restriction enzymes and thelike, are widely known in the art and commercially available from suchvendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega,U. S. Biochemicals, New England Nuclear, and a number of other sources.The recombinant nucleic acid sequences used to produce fusion proteinsof the present invention may be derived from natural or syntheticsequences. Many natural gene sequences are obtainable from various cDNAor from genomic libraries using appropriate probes. See, GenBank,National Institutes of Health.

[0071] As used herein, a “portion” of the MMSC2 locus or region orallele is defined as having a minimal size of at least about eightnucleotides, or preferably about 15 nucleotides, or more preferably atleast about 25 nucleotides, and may have a minimal size of at leastabout 40 nucleotides. This definition includes all sizes in the range of8-40 nucleotides as well as greater than 40 nucleotides. Thus, thisdefinition includes nucleic acids of 8, 12, 15, 20, 25, 40, 60, 80, 100,200, 300, 400, 500 nucleotides, or nucleic acids having any number ofnucleotides within these values (e.g., 9, 10, 11, 16, 23, 30, 38, 50,72, 121, etc, nucleotides), or nucleic acids having more than 500nucleotides, or any number of nucleotides between 500 and the numbershown in SEQ ID NO: 2. The present invention includes all novel nucleicacids having at least 8 nucleotides derived from SEQ ID NO: 2, itscomplement or functionally equivalent nucleic acid sequences. Thepresent invention does not include nucleic acids which exist in theprior art. That is, the present invention includes all nucleic acidshaving at least 8 nucleotides derived from SEQ ID NO: 2 with the provisothat it does not include nucleic acids existing in the prior art.

[0072] “MMSC2 protein” or “MMSC2 polypeptide” refers to a protein orpolypeptide encoded by the MMSC2 locus, variants or fragments thereof.The term “polypeptide” refers to a polymer of amino acids and itsequivalent and does not refer to a specific length of the product; thus,peptides, oligopeptides and proteins are included within the definitionof a polypeptide. This term also does not refer to, or excludemodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations, and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),polypeptides with substituted linkages as well as other modificationsknown in the art, both naturally and non-naturally occurring.

[0073] Ordinarily, such polypeptides will be at least about 50%homologous to the native MMSC2 sequence, preferably in excess of about90%, and more preferably at least about 95% homologous. Also includedare proteins encoded by DNA which hybridize under high or low stringencyconditions, to MMSC2-encoding nucleic acids and closely relatedpolypeptides or proteins retrieved by antisera to the MMSC2 protein(s).

[0074] The MMSC2 polypeptide may be that shown in SEQ ID NO: 3 which maybe in isolated and/or purified form, free or substantially free ofmaterial with which it is naturally associated. The polypeptide may, ifproduced by expression in a prokaryotic cell or produced synthetically,lack native post-translational processing, such as glycosylation.Alternatively, the present invention is also directed to polypeptideswhich are sequence variants, alleles or derivatives of the MMSC2polypeptide. Such polypeptides may have an amino acid sequence whichdiffers from that set forth in SEQ ID NO: 3 by one or more of addition,substitution, deletion or insertion of one or more amino acids.Preferred such polypeptides have MMSC2 function.

[0075] Substitutional variants typically contain the exchange of oneamino acid for another at one or more sites within the protein, and maybe designed to modulate one or more properties of the polypeptide, suchas stability against proteolytic cleavage, without the loss of otherfunctions or properties. Amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.Preferred substitutions are ones which are conservative, that is, oneamino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and typicallyinclude substitutions within the following groups: glycine, alanine;valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and tyrosine,phenylalanine.

[0076] Certain amino acids may be substituted for other amino acids in aprotein structure without appreciable loss of interactive bindingcapacity with structures such as, for example, antigen-binding regionsof antibodies or binding sites on substrate molecules or binding siteson proteins interacting with the MMSC2 polypeptide. Since it is theinteractive capacity and nature of a protein which defines thatprotein's biological functional activity, certain amino acidsubstitutions can be made in a protein sequence, and its underlying DNAcoding sequence, and nevertheless obtain a protein with like properties.In making such changes, the hydrophathic index of amino acids may beconsidered. The importance of the hydrophobic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982). Alternatively, thesubstitution of like amino acids can be made effectively on the basis ofhydrophilicity. The importance of hydrophilicity in conferringinteractive biological function of a protein is generally understood inthe art (U.S. Pat. No. 4,554,101). The use of the hydrophobic index orhydrophilicity in designing polypeptides is further discussed in U.S.Pat. No. 5,691,198.

[0077] The length of polypeptide sequences compared for homology willgenerally be at least about 16 amino acids, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues.

[0078] “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. For instance, a promoter is operablylinked to a coding sequence if the promoter affects its transcription orexpression.

[0079] The term “peptide mimetic” or “mimetic” is intended to refer to asubstance which has the essential biological activity of the MMSC2polypeptide. A peptide mimetic may be a peptide-containing molecule thatmimics elements of protein secondary structure (Johnson et al., 1993).The underlying rationale behind the use of peptide mimetics is that thepeptide backbone of proteins exists chiefly to orient amino acid sidechains in such a way as to facilitate molecular interactions, such asthose of antibody and antigen, enzyme and substrate or scaffoldingproteins. A peptide mimetic is designed to permit molecular interactionssimilar to the natural molecule. A mimetic may not be a peptide at all,but it will retain the essential biological activity of natural MMSC2polypeptide.

[0080] “Probes”. Polynucleotide polymorphisms associated with MMSC2alleles which predispose to cancer are detected by hybridization with apolynucleotide probe which forms a stable hybrid with that of the targetsequence, under highly stringent to moderately stringent hybridizationand wash conditions. If it is expected that the probes will be perfectlycomplementary to the target sequence, high stringency conditions will beused. Hybridization stringency may be lessened if some mismatching isexpected, for example, if variants are expected with the result that theprobe will not be completely complementary. Conditions are chosen whichrule out nonspecific/adventitious bindings, that is, which minimizenoise. (It should be noted that throughout this disclosure, if it issimply stated that “stringent” conditions are used that is meant to beread as “high stringency” conditions are used.) Since such indicationsidentify neutral DNA polymorphisms as well as mutations, theseindications need further analysis to demonstrate detection of a MMSC2susceptibility allele.

[0081] Probes for MMSC2 alleles may be derived from the sequences of theMMSC2 region, its cDNA, functionally equivalent sequences, or thecomplements thereof. The probes may be of any suitable length, whichspan all or a portion of the MMSC2 region, and which allow specifichybridization to the region. If the target sequence contains a sequenceidentical to that of the probe, the probes may be short, e.g., in therange of about 8-30 base pairs, since the hybrid will be relativelystable under even highly stringent conditions. If some degree ofmismatch is expected with the probe, i.e., if it is suspected that theprobe will hybridize to a variant region, a longer probe may be employedwhich hybridizes to the target sequence with the requisite specificity.

[0082] The probes will include an isolated polynucleotide attached to alabel or reporter molecule and may be used to isolate otherpolynucleotide sequences, having sequence similarity by standardmethods. For techniques for preparing and labeling probes see, e.g.,Sambrook et al., 1989 or Ausubel et al., 1992. Other similarpolynucleotides may be selected by using homologous polynucleotides.Alternatively, polynucleotides encoding these or similar polypeptidesmay be synthesized or selected by use of the redundancy in the geneticcode. Various codon substitutions may be introduced, e.g., by silentchanges (thereby producing various restriction sites) or to optimizeexpression for a particular system. Mutations may be introduced tomodify the properties of the polypeptide, perhaps to change thepolypeptide degradation or turnover rate.

[0083] Probes comprising synthetic oligonucleotides or otherpolynucleotides of the present invention may be derived from naturallyoccurring or recombinant single- or double-stranded polynucleotides, orbe chemically synthesized. Probes may also be labeled by nicktranslation, Klenow fill-in reaction, or other methods known in the art.

[0084] Portions of the polynucleotide sequence having at least abouteight nucleotides, usually at least about 15 nucleotides, and fewer thanabout 9 kb, usually fewer than about 1.0 kb, from a polynucleotidesequence encoding MMSC2 are preferred as probes. This definitiontherefore includes probes of sizes 8 nucleotides through 9000nucleotides. Thus, this definition includes probes of 8, 12, 15, 20, 25,40, 60, 80, 100, 200, 300, 400, 500 nucleotides, or probes having anynumber of nucleotides within these values (e.g., 9, 10, 11, 16, 23, 30,38, 50, 72, 121, etc, nucleotides), or probes having more than 500nucleotides, or any number of nucleotides between 500 and the numbershown in SEQ ID NO: 2. The probes may also be used to determine whethermRNA encoding MMSC2 is present in a cell or tissue. The presentinvention includes all novel probes having at least 8 nucleotidesderived from SEQ ID NO: 2, its complement or functionally equivalentnucleic acid sequences. The present invention does not include probeswhich exist in the prior art. That is, the present invention includesall probes having at least 8 nucleotides derived from SEQ ID NO: 2 withthe proviso that it does not include probes existing in the prior art.

[0085] Similar considerations and nucleotide lengths are also applicableto primers which may be used for the amplification of all or part of theMMSC2 gene. Thus, a definition for primers includes primers of 8, 12,15, 20, 25, 40, 60, 80, 100, 200, 300, 400, 500 nucleotides, or primershaving any number of nucleotides within these values (e.g., 9, 10, 11,16, 23, 30, 38, 50, 72, 121, etc, nucleotides), or primers having morethan 500 nucleotides, or any number of nucleotides between 500 and 9000.The primers may also be used to determine whether mRNA encoding MMSC2 ispresent in a cell or tissue. The present invention includes all novelprimers having at least 8 nucleotides derived from the MMSC2 locus foramplifying the MMSC2 gene, its complement or functionally equivalentnucleic acid sequences. The present invention does not include primerswhich exist in the prior art. That is, the present invention includesall primers having at least 8 nucleotides with the proviso that it doesnot include primers existing in the prior art.

[0086] “Protein modifications or fragments” are provided by the presentinvention for MMSC2 polypeptides or fragments thereof which aresubstantially homologous to primary structural sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate unusual amino acids. Suchmodifications include, for example, acetylation, carboxylation,phosphorylation, glycosylation, ubiquitination, labeling, e.g., withradionuclides, and various enzymatic modifications, as will be readilyappreciated by those well skilled in the art. A variety of methods forlabeling polypeptides and of substituents or labels useful for suchpurposes are well known in the art, and include radioactive isotopessuch as ³²P, ligands which bind to labeled antiligands (e.g.,antibodies), fluorophores, chemiluminescent agents, enzymes, andantiligands which can serve as specific binding pair members for alabeled ligand. The choice of label depends on the sensitivity required,ease of conjugation with the primer, stability requirements, andavailable instrumentation. Methods of labeling polypeptides are wellknown in the art. See Sambrook et al., 1989 or Ausubel et al., 1992.

[0087] Besides substantially full-length polypeptides, the presentinvention provides for biologically active fragments of thepolypeptides. Significant biological activities include ligand-binding,immunological activity and other biological activities characteristic ofMMSC2 polypeptides. Immunological activities include both immunogenicfunction in a target immune system, as well as sharing of immunologicalepitopes for binding, serving as either a competitor or substituteantigen for an epitope of the MMSC2 protein. As used herein, “epitope”refers to an antigenic determinant of a polypeptide. An epitope couldcomprise three amino acids in a spatial conformation which is unique tothe epitope. Generally, an epitope consists of at least five such aminoacids, and more usually consists of at least 8-10 such amino acids.Methods of determining the spatial conformation of such amino acids areknown in the art.

[0088] For immunological purposes, tandem-repeat polypeptide segmentsmay be used as immunogens, thereby producing highly antigenic proteins.Alternatively, such polypeptides will serve as highly efficientcompetitors for specific binding. Production of antibodies specific forMMSC2 polypeptides or fragments thereof is described below.

[0089] The present invention also provides for fusion polypeptides,comprising MMSC2 polypeptides and fragments. Homologous polypeptides maybe fusions between two or more MMSC2 polypeptide sequences or betweenthe sequences of MMSC2 and a related protein. Likewise, heterologousfusions may be constructed which would exhibit a combination ofproperties or activities of the derivative proteins. For example,ligand-binding or other domains may be “swapped” between different newfusion polypeptides or fragments. Such homologous or heterologous fusionpolypeptides may display, for example, altered strength or specificityof binding. Fusion partners include immunoglobulins, bacterialβ-galactosidase, trpE, protein A, β-lactamase, α-amylase, alcoholdehydrogenase and yeast alpha mating factor. See Godowski et al., 1988.

[0090] Fusion proteins will typically be made by either recombinantnucleic acid methods, as described below, or may be chemicallysynthesized. Techniques for the synthesis of polypeptides are described,for example, in Merrifield, 1963.

[0091] “Protein purification” refers to various methods for theisolation of the MMSC2 polypeptides from other biological material, suchas from cells transformed with recombinant nucleic acids encoding MMSC2,and are well known in the art. For example, such polypeptides may bepurified by immunoaffinity chromatography employing, e.g., theantibodies provided by the present invention. Various methods of proteinpurification are well known in the art, and include those described inDeutscher, 1990 and Scopes, 1982.

[0092] The terms “isolated”, “substantially pure”, and “substantiallyhomogeneous” are used interchangeably to describe a protein orpolypeptide which has been separated from components which accompany itin its natural state. A monomeric protein is substantially pure when atleast about 60 to 75% of a sample exhibits a single polypeptidesequence. A substantially pure protein will typically comprise about 60to 90% W/W of a protein sample, more usually about 95%, and preferablywill be over about 99% pure. Protein purity or homogeneity may beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein sample, followed byvisualizing a single polypeptide band upon staining the gel. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art which are utilized for purification.

[0093] A MMSC2 protein is substantially free of naturally associatedcomponents when it is separated from the native contaminants whichaccompany it in its natural state. Thus, a polypeptide which ischemically synthesized or synthesized in a cellular system differentfrom the cell from which it naturally originates will be substantiallyfree from its naturally associated components. A protein may also berendered substantially free of naturally associated components byisolation, using protein purification techniques well known in the art.

[0094] A polypeptide produced as an expression product of an isolatedand manipulated genetic sequence is an “isolated polypeptide,” as usedherein, even if expressed in a homologous cell type. Synthetically madeforms or molecules expressed by heterologous cells are inherentlyisolated molecules.

[0095] “Recombinant nucleic acid” is a nucleic acid which is notnaturally occurring, or which is made by the artificial combination oftwo otherwise separated segments of sequence. This artificialcombination is often accomplished by either chemical synthesis means, orby the artificial manipulation of isolated segments of nucleic acids,e.g., by genetic engineering techniques. Such is usually done to replacea codon with a redundant codon encoding the same or a conservative aminoacid, while typically introducing or removing a sequence recognitionsite. Alternatively, it is performed to join together nucleic acidsegments of desired functions to generate a desired combination offunctions.

[0096] “Regulatory sequences” refers to those sequences normally within100 kb of the coding region of a locus, but they may also be moredistant from the coding region, which affect the expression of the gene(including transcription of the gene, and translation, splicing,stability or the like of the messenger RNA).

[0097] “Substantial homology or similarity”. A nucleic acid or fragmentthereof is “substantially homologous” (“or substantially similar”) toanother if, when optimally aligned (with appropriate nucleotideinsertions or deletions) with the other nucleic acid (or itscomplementary strand), there is nucleotide sequence identity in at leastabout 60% of the nucleotide bases, usually at least about 70%, moreusually at least about 80%, preferably at least about 90%, and morepreferably at least about 95-98% of the nucleotide bases.

[0098] Alternatively, substantial homology or (similarity) exists when anucleic acid or fragment thereof will hybridize to another nucleic acid(or a complementary strand thereof) under selective hybridizationconditions, to a strand, or to its complement. Selectivity ofhybridization exists when hybridization which is substantially moreselective than total lack of specificity occurs. Typically, selectivehybridization will occur when there is at least about 55% homology overa stretch of at least about 14 nucleotides, preferably at least about65%, more preferably at least about 75%, and most preferably at leastabout 90%. See, Kanehisa, 1984. The length of homology comparison, asdescribed, may be over longer stretches, and in certain embodiments willoften be over a stretch of at least about nine nucleotides, usually atleast about 20 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about32 nucleotides, and preferably at least about 36 or more nucleotides.

[0099] Nucleic acid hybridization will be affected by such conditions assalt concentration, temperature, or organic solvents, in addition to thebase composition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess of30° C., typically in excess of 37° C., and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1000 mM,typically less than 500 mM, and preferably less than 200 mM. However,the combination of parameters is much more important than the measure ofany single parameter. The stringency conditions are dependent on thelength of the nucleic acid and the base composition of the nucleic acidand can be determined by techniques well known in the art. See, e.g.,Wetmur and Davidson, 1968.

[0100] Probe sequences may also hybridize specifically to duplex DNAunder certain conditions to form triplex or other higher order DNAcomplexes. The preparation of such probes and suitable hybridizationconditions are well known in the art.

[0101] The terms “substantial homology” or “substantial identity”, whenreferring to polypeptides, indicate that the polypeptide or protein inquestion exhibits at least about 30% identity with an entirenaturally-occurring protein or a portion thereof, usually at least about70% identity, more usually at least about 80% identity, preferably atleast about 90% identity, and more preferably at least about 95%identity.

[0102] Homology, for polypeptides, is typically measured using sequenceanalysis software. See, e.g., the Sequence Analysis Software Package ofthe Genetics Computer Group, University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

[0103] “Substantially similar function” refers to the function of amodified nucleic acid or a modified protein, with reference to thewild-type MMSC2 nucleic acid or wild-type MMSC2 polypeptide. Themodified polypeptide will be substantially homologous to the wild-typeMMSC2 polypeptide and will have substantially the same function. Themodified polypeptide may have an altered amino acid sequence and/or maycontain modified amino acids. In addition to the similarity of function,the modified polypeptide may have other useful properties, such as alonger half-life. The similarity of function (activity) of the modifiedpolypeptide may be substantially the same as the activity of thewild-type MMSC2 polypeptide. Alternatively, the similarity of function(activity) of the modified polypeptide may be higher than the activityof the wild-type MMSC2 polypeptide. The modified polypeptide issynthesized using conventional techniques, or is encoded by a modifiednucleic acid and produced using conventional techniques. The modifiednucleic acid is prepared by conventional techniques. A nucleic acid witha function substantially similar to the wild-type MMSC2 gene functionproduces the modified protein described above.

[0104] A polypeptide “fragment,” “portion” or “segment” is a stretch ofamino acid residues of at least about five to seven contiguous aminoacids, often at least about seven to nine contiguous amino acids,typically at least about nine to 13 contiguous amino acids and, mostpreferably, at least about 20 to 30 or more contiguous amino acids.

[0105] The polypeptides of the present invention, if soluble, may becoupled to a solid-phase support, e.g., nitrocellulose, nylon, columnpacking materials (e.g., Sepharose beads), magnetic beads, glass wool,plastic, metal, polymer gels, cells, or other substrates. Such supportsmay take the form, for example, of beads, wells, dipsticks, ormembranes.

[0106] “Target region” refers to a region of the nucleic acid which isamplified and/or detected. The term “target sequence” refers to asequence with which a probe or primer will form a stable hybrid underdesired conditions.

[0107] The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, and immunology. See, e.g.,Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al., 1992;Glover, 1985; Anand, 1992; Guthrie and Fink, 1991. A general discussionof techniques and materials for human gene mapping, including mapping ofhuman chromosome 1, is provided, e.g., in White and Lalouel, 1988.

[0108] Preparation of Recombinant or Chemically Synthesized NucleicAcids; Vectors, Transformation, Host Cells

[0109] Large amounts of the polynucleotides of the present invention maybe produced by replication in a suitable host cell. Natural or syntheticpolynucleotide fragments coding for a desired fragment will beincorporated into recombinant polynucleotide constructs, usually DNAconstructs, capable of introduction into and replication in aprokaryotic or eukaryotic cell. Usually the polynucleotide constructswill be suitable for replication in a unicellular host, such as yeast orbacteria, but may also be intended for introduction to (with and withoutintegration within the genome) cultured mammalian, plant, insect orother eukaryotic cell lines. The purification of nucleic acids producedby the methods of the present invention are described, e.g., in Sambrooket al., 1989 or Ausubel et al., 1992.

[0110] The polynucleotides of the present invention may also be producedby chemical synthesis, e.g., by the phosphoramidite method described byBeaucage and Carruthers, 1981 or the triester method according toMatteucci and Caruthers, 1981, and may be performed on commercial,automated oligonucleotide synthesizers. A double-stranded fragment maybe obtained from the single-stranded product of chemical synthesiseither by synthesizing the complementary strand and annealing the strandtogether under appropriate conditions or by adding the complementarystrand using DNA polymerase with an appropriate primer sequence.

[0111] Polynucleotide constructs prepared for introduction into aprokaryotic or eukaryotic host may comprise a replication systemrecognized by the host, including the intended polynucleotide fragmentencoding the desired polypeptide, and will preferably also includetranscription and translational initiation regulatory sequences operablylinked to the polypeptide encoding segment. Expression vectors mayinclude, for example, an origin of replication or autonomouslyreplicating sequence (ARS) and expression control sequences, a promoter,an enhancer and necessary processing information sites, such asribosome-binding sites, RNA splice sites, polyadenylation sites,transcriptional terminator sequences, and mRNA stabilizing sequences.Such vectors may be prepared by means of standard recombinant techniqueswell known in the art and discussed, for example, in Sambrook et al.,1989 or Ausubel et al., 199.

[0112] An appropriate promoter and other necessary vector sequences willbe selected so as to be functional in the host, and may include, whenappropriate, those naturally associated with the MMSC2 gene. Examples ofworkable combinations of cell lines and expression vectors are describedin Sambrook et al., 1989 or Ausubel et al., 1992; see also, e.g.,Metzger et al., 1988. Many useful vectors are known in the art and maybe obtained from such vendors as Stratagene, New England Biolabs,Promega Biotech, and others. Promoters such as the trp, lac and phagepromoters, tRNA promoters and glycolytic enzyme promoters may be used inprokaryotic hosts. Useful yeast promoters include promoter regions formetallothionein, 3-phosphoglycerate kinase or other glycolytic enzymessuch as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymesresponsible for maltose and galactose utilization, and others. Vectorsand promoters suitable for use in yeast expression are further describedin Hitzeman et al. (EP 73,675A). Appropriate non-native mammalianpromoters might include the early and late promoters from SV40 (Fiers etal., 1978) or promoters derived from murine Molony leukemia virus, mousetumor virus, avian sarcoma viruses, adenovirus II, bovine papillomavirus or polyoma. Insect promoters may be derived from baculovirus. Inaddition, the construct may be joined to an amplifiable gene (e.g.,DHFR) so that multiple copies of the gene may be made. For appropriateenhancer and other expression control sequences, see also Enhancers andEukaryotic Gene Expression, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1983). See also, e.g., U.S. Pat. No. 5,691,198.

[0113] While such expression vectors may replicate autonomously, theymay also replicate by being inserted into the genome of the host cell,by methods well known in the art.

[0114] Expression and cloning vectors will likely contain a selectablemarker, a gene encoding a protein necessary for survival or growth of ahost cell transformed with the vector. The presence of this gene ensuresgrowth of only those host cells which express the inserts. Typicalselection genes encode proteins that a) confer resistance to antibioticsor other toxic substances, e.g. ampicillin, neomycin, methotrexate,etc., b) complement auxotrophic deficiencies, or c) supply criticalnutrients not available from complex media, e.g., the gene encodingD-alanine racemase for Bacilli. The choice of the proper selectablemarker will depend on the host cell, and appropriate markers fordifferent hosts are well known in the art.

[0115] The vectors containing the nucleic acids of interest can betranscribed in vitro, and the resulting RNA introduced into the hostcell by well-known methods, e.g., by injection (see, Kubo et al., 1988),or the vectors can be introduced directly into host cells by methodswell known in the art, which vary depending on the type of cellularhost, including electroporation; transfection employing calciumchloride, rubidium chloride calcium phosphate, DEAE-dextran, or othersubstances; microprojectile bombardment; lipofection; infection (wherethe vector is an infectious agent, such as a retroviral genome); andother methods. See generally, Sambrook et al., 1989 and Ausubel et al.,1992. The introduction of the polynucleotides into the host cell by anymethod known in the art, including, inter alia, those described aboveand in U.S. Pat. No. 5,691,198, will be referred to herein as“transformation.” The cells into which have been introduced nucleicacids described above are meant to also include the progeny of suchcells.

[0116] Large quantities of the nucleic acids and polypeptides of thepresent invention may be prepared by expressing the MMSC2 nucleic acidor portions thereof in vectors or other expression vehicles incompatible prokaryotic or eukaryotic host cells. The most commonly usedprokaryotic hosts are strains of Escherichia coli, although otherprokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.

[0117] Mammalian or other eukaryotic host cells, such as those of yeast,filamentous fungi, plant, insect, or amphibian or avian species, mayalso be useful for production of the proteins of the present invention.Propagation of mammalian cells in culture is per se well known. See,Jakoby and Pastan (eds.), 1979. Examples of commonly used mammalian hostcell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells,and WI38, BHK, and COS cell lines. An example of a commonly used insectcell line is SF9. However, it will be appreciated by the skilledpractitioner that other cell lines may be appropriate, e.g., to providehigher expression, desirable glycosylation patterns, or other features.

[0118] Clones are selected by using markers depending on the mode of thevector construction. The marker may be on the same or a different DNAmolecule, preferably the same DNA molecule. In prokaryotic hosts, thetransformant may be selected, e.g., by resistance to ampicillin,tetracycline or other antibiotics. Production of a particular productbased on temperature sensitivity may also serve as an appropriatemarker.

[0119] Prokaryotic or eukaryotic cells transformed with thepolynucleotides of the present invention will be useful not only for theproduction of the nucleic acids and polypeptides of the presentinvention, but also, for example, in studying the characteristics ofMMSC2 polypeptide.

[0120] The probes and primers based on the MMSC2 gene sequence disclosedherein are used to identify homologous MMSC2 gene sequences and proteinsin other species. These gene sequences and proteins are used in thediagnostic/prognostic, therapeutic and drug screening methods describedherein for the species from which they have been isolated.

[0121] Methods of Use: Drug Screening

[0122] This invention is particularly useful for screening compounds byusing the MMSC2 polypeptide or binding fragment thereof in any of avariety of drug screening techniques, such as those described herein andin published PCT application WO 97/02048. Since MMSC2 acts as a scaffoldthat binds to MMAC1, the phosphatase substrate(s) and the (probablyoncogene) tyrosine kinase(s), a valuable drug candidate will be a drugthat can prevent binding of either the substrate(s) or the tyrosinekinase(s) to MMSC2.

[0123] The MMSC2 polypeptide or fragment employed in such a test mayeither be free in solution, affixed to a solid support, or borne on acell surface. One method of drug screening utilizes eukaryotic orprocaryotic host cells which are stably transformed with recombinantpolynucleotides expressing the polypeptide or fragment, preferably incompetitive binding assays. Such cells, either in viable or fixed form,can be used for standard binding assays. One may measure, for example,for the formation of complexes between a MMSC2 polypeptide or fragmentand the agent being tested, or examine the degree to which the formationof a complex between a MMSC2 polypeptide or fragment and a known ligand,e.g., MMAC1, is aided or interfered with by the agent being tested.

[0124] Thus, the present invention provides methods of screening fordrugs comprising contacting such an agent with a MMSC2 polypeptide orfragment thereof and assaying (i) for the presence of a complex betweenthe agent and the MMSC2 polypeptide or fragment, or (ii) for thepresence of a complex between the MMSC2 polypeptide or fragment and aligand, by methods well known in the art. In such competitive bindingassays the MMSC2 polypeptide or fragment is typically labeled. FreeMMSC2 polypeptide or fragment is separated from that present in aprotein:protein complex, and the amount of free (i.e., uncomplexed)label is a measure of the binding of the agent being tested to MMSC2 orits interference with or promotion of MMSC2: ligand binding,respectively. One may also measure the amount of bound, rather thanfree, MMSC2. It is also possible to label the ligand rather than theMMSC2 and to measure the amount of ligand binding to MMSC2 in thepresence and in the absence of the drug being tested.

[0125] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to the MMSC2polypeptides and is described in detail in Geysen (published PCTapplication WO 84/03564). Briefly stated, large numbers of differentsmall peptide test compounds are synthesized on a solid substrate, suchas plastic pins or some other surface. The peptide test compounds arereacted with MMSC2 polypeptide and washed. Bound MMSC2 polypeptide isthen detected by methods well known in the art.

[0126] Purified MMSC2 can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to the polypeptide can be used to capture antibodies toimmobilize the MMSC2 polypeptide on the solid phase.

[0127] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable ofspecifically binding the MMSC2 polypeptide compete with a test compoundfor binding to the MMSC2 polypeptide or fragments thereof. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants of the MMSC2polypeptide.

[0128] The above screening methods are not limited to assays employingonly MMSC2 but are also applicable to studying MMSC2-protein complexes,e.g., the complex which occurs between MMSC2 and MMAC1. The effect ofdrugs on the activity of this complex, especially when either the MMSC2or the MMSC2 binding protein (e.g., MMAC1) contains a mutation, isanalyzed.

[0129] In accordance with these methods, the following assays areexamples of assays which can be used for screening for drug candidates.

[0130] A mutant MMSC2 (per se or as part of a fusion protein) is mixedwith a wild-type protein (per se or as part of a fusion protein) towhich wild-type MMSC2 binds. This mixing is performed in both thepresence of a drug and the absence of the drug, and the amount ofbinding of the mutant MMSC2 with the wild-type protein is measured. Ifthe amount of the binding is more in the presence of said drug than inthe absence of said drug, the drug is a drug candidate for treatingcancer resulting from a mutation in MMSC2. This assay is useful wherethe wild-type protein is a tumor suppressor, such as MMAC1.

[0131] A wild-type MMSC2 (per se or as part of a fusion protein) ismixed with a wild-type protein (per se or as part of a fusion protein)to which wild-type MMSC2 binds. This mixing is performed in both thepresence of a drug and the absence of the drug, and the amount ofbinding of the wild-type MMSC2 with the wild-type protein is measured.If the amount of the binding is more in the presence of said drug thanin the absence of said drug, the drug is a drug candidate for treatingcancer resulting from a mutation in MMSC2. This assay is useful wherethe wild-type protein is a tumor suppressor, such as MMAC1.

[0132] A mutant MMSC2 (per se or as part of a fusion protein) is mixedwith a wild-type protein (per se or as part of a fusion protein) towhich wild-type MMSC2 binds. This mixing is performed in both thepresence of a drug and the absence of the drug, and the amount ofbinding of the mutant MMSC2 with the wild-type protein is measured. Ifthe amount of the binding is less in the presence of said drug than inthe absence of said drug, the drug is a drug candidate for treatingcancer resulting from a mutation in MMSC2. This assay is useful if theprotein is an oncoprotein or a substrate of the oncoprotein.

[0133] A wild-type MMSC2 (per se or as part of a fusion protein) ismixed with a wild-type protein (per se or as part of a fusion protein)to which wild-type MMSC2 binds. This mixing is performed in both thepresence of a drug and the absence of the drug, and the amount ofbinding of the wild-type MMSC2 with the wild-type protein is measured.If the amount of the binding is less in the presence of said drug thanin the absence of said drug, the drug is a drug candidate for treatingcancer resulting from a mutation in MMSC2 or a cancer resulting from amutation in MMAC1. This assay is useful if the protein is an oncoproteinor a substrate of the oncoprotein.

[0134] A mutant protein, which as a wild-type protein binds to MMSC2(per se or as part of a fusion protein) is mixed with a wild-type MMSC2(per se or as part of a fusion protein). This mixing is performed inboth the presence of a drug and the absence of the drug, and the amountof binding of the mutant protein with the wild-type MMSC2 is measured.If the amount of the binding is less in the presence of said drug thanin the absence of said drug, the drug is a drug candidate for treatingcancer resulting from a mutation in the gene encoding the protein.

[0135] The polypeptide of the invention may also be used for screeningcompounds developed as a result of combinatorial library technology.Combinatorial library technology provides an efficient way of testing apotential vast number of different substances for ability to modulateactivity of a polypeptide. Such libraries and their use are known in theart. The use of peptide libraries is preferred. See, for example, WO97/02048.

[0136] Briefly, a method of screening for a substance which modulatesactivity of a polypeptide may include contacting one or more testsubstances with the polypeptide in a suitable reaction medium, testingthe activity of the treated polypeptide and comparing that activity withthe activity of the polypeptide in comparable reaction medium untreatedwith the test substance or substances. A difference in activity betweenthe treated and untreated polypeptides is indicative of a modulatingeffect of the relevant test substance or substances.

[0137] Prior to or as well as being screened for modulation of activity,test substances may be screened for ability to interact with thepolypeptide, e.g., in a yeast two-hybrid system (e.g., Bartel et al.,1993). This system may be used as a coarse screen prior to testing asubstance for actual ability to modulate activity of the polypeptide.Alternatively, the screen could be used to screen test substances forbinding to a MMSC2 specific binding partner, such as MMAC1, or to findmimetics of the MMSC2 polypeptide.

[0138] Following identification of a substance which modulates oraffects polypeptide activity, the substance may be investigated further.Furthermore, it may be manufactured and/or used in preparation, i.e.,manufacture or formulation, or a composition such as a medicament,pharmaceutical composition or drug. These may be administered toindividuals.

[0139] Thus, the present invention extends in various aspects not onlyto a substance identified using a nucleic acid molecule as a modulatorof polypeptide activity, in accordance with what is disclosed herein,but also a pharmaceutical composition, medicament, drug or othercomposition comprising such a substance, a method comprisingadministration of such a composition comprising such a substance, amethod comprising administration of such a composition to a patient,e.g., for treatment (which may include preventative treatment) ofcancer, use of such a substance in the manufacture of a composition foradministration, e.g., for treatment of cancer, and a method of making apharmaceutical composition comprising admixing such a substance with apharmaceutically acceptable excipient, vehicle or carrier, andoptionally other ingredients.

[0140] A substance identified using as a modulator of polypeptidefunction may be peptide or non-peptide in nature. Non-peptide “smallmolecules” are often preferred for many in vivo pharmaceutical uses.Accordingly, a mimetic or mimic of the substance (particularly if apeptide) may be designed for pharmaceutical use.

[0141] The designing of mimetics to a known pharmaceutically activecompound is a known approach to the development of pharmaceuticals basedon a “lead” compound. This might be desirable where the active compoundis difficult or expensive to synthesize or where it is unsuitable for aparticular method of administration, e.g., peptides are unsuitableactive agents for oral compositions as they tend to be quickly degradedby proteases in the alimentary canal. Mimetic design, synthesis andtesting is generally used to avoid randomly screening large numbers ofmolecules for a target property.

[0142] There are several steps commonly taken in the design of a mimeticfrom a compound having a given target property. First, the particularparts of the compound that are critical and/or important in determiningthe target property are determined. In the case of a peptide, this canbe done by systematically varying the mino acid residues in the peptide,e.g., by substituting each residue in turn. Alanine scans of peptide arecommonly used to refine such peptide motifs. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

[0143] Once the pharmacophore has been found, its structure is modeledaccording to its physical properties, e.g., stereochemistry, bonding,size and/or charge, using data from a range of sources, e.g.,spectroscopic techniques, x-ray diffraction data and NMR. Computationalanalysis, similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modeling process.

[0144] In a variant of this approach, the three-dimensional structure ofthe ligand and its binding partner are modeled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the design of themimetic.

[0145] A template molecule is then selected onto which chemical groupswhich mimic the pharmacophore can be grafted. The template molecule andthe chemical groups grafted onto it can conveniently be selected so thatthe mimetic is easy to synthesize, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. Alternatively, where the mimetic ispeptide-based, further stability can be achieved by cyclizing thepeptide, increasing its rigidity. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimization ormodification can then be carried out to arrive at one or more ifinalmimetics for in vivo or clinical testing.

[0146] Methods of Use: Nucleic Acid Diagnosis and Diagnostic Kits

[0147] In order to detect the presence of a MMSC2 allele predisposing anindividual to cancer, a biological sample such as blood is prepared andanalyzed for the presence or absence of susceptibility alleles of MMSC2.In order to detect the presence of cancer or as a prognostic indicator,a biological sample is prepared and analyzed for the presence or absenceof mutant alleles of MMSC2. Results of these tests and interpretiveinformation are returned to the health care provider for communicationto the tested individual. Such diagnoses may be performed by diagnosticlaboratories, or, alternatively, diagnostic kits are manufactured andsold to health care providers or to private individuals forself-diagnosis.

[0148] Initially, the screening method involves amplification of therelevant MMSC2 sequences. In another preferred embodiment of theinvention, the screening method involves a non-PCR based strategy. Suchscreening methods include two-step label amplification methodologiesthat are well known in the art. Both PCR and non-PCR based screeningstrategies can detect target sequences with a high level of sensitivity.

[0149] The most popular method used today is target amplification. Here,the target nucleic acid sequence is amplified with polymerases. Oneparticularly preferred method using polymerase-driven amplification isthe polymerase chain reaction (PCR). The polymerase chain reaction andother polymerase-driven amplification assays can achieve over amillion-fold increase in copy number through the use ofpolymerase-driven amplification cycles. Once amplified, the resultingnucleic acid can be sequenced or used as a substrate for DNA probes.

[0150] When the probes are used to detect the presence of the targetsequences the biological sample to be analyzed, such as blood or serum,may be treated, if desired, to extract the nucleic acids. The samplenucleic acid may be prepared in various ways to facilitate detection ofthe target sequence, e.g., denaturation, restriction digestion,electrophoresis or dot blotting. The targeted region of the analytenucleic acid usually must be at least partially single-stranded to formhybrids with the targeting sequence of the probe. If the sequence isnaturally single-stranded, denaturation will not be required. However,if the sequence is double-stranded, the sequence will probably need tobe denatured. Denaturation can be carried out by various techniquesknown in the art.

[0151] Analyte nucleic acid and probe are incubated under conditionswhich promote stable hybrid formation of the target sequence in theprobe with the putative targeted sequence in the analyte. The region ofthe probes which is used to bind to the analyte can be made completelycomplementary to the targeted region for MMSC2. Therefore, highstringency conditions are desirable in order to prevent false positives.However, conditions of high stringency are used only if the probes arecomplementary to regions of the chromosome which are unique in thegenome. The stringency of hybridization is determined by a number offactors during hybridization and during the washing procedure, includingtemperature, ionic strength, base composition, probe length, andconcentration of formamide. These factors are outlined in, for example,Maniatis et al., 1982 and Sambrook et al., 1989. Under certaincircumstances, the formation of higher order hybrids, such as triplexes,quadraplexes, etc., may be desired to provide the means of detectingtarget sequences.

[0152] Detection, if any, of the resulting hybrid is usuallyaccomplished by the use of labeled probes. Alternatively, the probe maybe unlabeled, but may be detectable by specific binding with a ligandwhich is labeled, either directly or indirectly. Suitable labels, andmethods for labeling probes and ligands are known in the art, andinclude, for example, radioactive labels which may be incorporated byknown methods (e.g., nick translation, random priming or kinasing),biotin, fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies, goldnanoparticles and the like. Variations of this basic scheme are known inthe art, and include those variations that facilitate separation of thehybrids to be detected from extraneous materials and/or that amplify thesignal from the labeled moiety. A number of these variations arereviewed in, e.g., Matthews and Kricka, 1988; Landegren et al., 1988;U.S. Pat. No. 4,868,105; and in EP 225,807A.

[0153] As noted above, non-PCR based screening assays are alsocontemplated in this invention. This procedure hybridizes a nucleic acidprobe (or an analog such as a methyl phosphonate backbone replacing thenormal phosphodiester), to the low level DNA target. This probe may havean enzyme covalently linked to the probe, such that the covalent linkagedoes not interfere with the specificity of the hybridization. Thisenzyme-probe-conjugate-target nucleic acid complex can then be isolatedaway from the free probe enzyme conjugate and a substrate is added forenzyme detection. Enzymatic activity is observed as a change in colordevelopment or luminescent output resulting in a 10³-10⁶ increase insensitivity. For an example relating to the preparation ofoligodeoxynucleotide-alkaline phosphatase conjugates and their use ashybridization probes, see Jablonski et al., 1986.

[0154] Two-step label amplification methodologies are known in the art.These assays work on the principle that a small ligand (such asdigoxigenin, biotin, or the like) is attached to a nucleic acid probecapable of specifically binding MMSC2. Allele specific probes are alsocontemplated within the scope of this example and exemplary allelespecific probes include probes encompassing the predisposing mutationsof this disclosure.

[0155] In one example, the small ligand attached to the nucleic acidprobe is specifically recognized by an antibody-enzyme conjugate. In oneembodiment of this example, digoxigenin is attached to the nucleic acidprobe. Hybridization is detected by an antibody-alkaline phosphataseconjugate which turns over a chemiluminescent substrate. For methods forlabeling nucleic acid probes according to this embodiment see Martin etal., 1990. In a second example, the small ligand is recognized by asecond ligand-enzyme conjugate that is capable of specificallycomplexing to the first ligand. A well known embodiment of this exampleis the biotin-avidin type of interactions. For methods for labelingnucleic acid probes and their use in biotin-avidin based assays seeRigby et al., 1977 and Nguyen et al., 1992.

[0156] It is also contemplated within the scope of this invention thatthe nucleic acid probe assays of this invention will employ a cocktailof nucleic acid probes capable of detecting MMSC2. Thus, in one exampleto detect the presence of MMSC2 in a cell sample, more than one probecomplementary to the gene is employed and in particular the number ofdifferent probes is alternatively two, three, or five different nucleicacid probe sequences. In another example, to detect the presence ofmutations in the MMSC2 gene sequence in a patient, more than one probecomplementary to these genes is employed where the cocktail includesprobes capable of binding to the allele-specific mutations identified inpopulations of patients with alterations in MMSC2. In this embodiment,any number of probes can be used, and will preferably include probescorresponding to the major gene mutations identified as predisposing anindividual to cancer.

[0157] Methods of Use: Peptide Diagnosis and Diagnostic Kits

[0158] The presence of cancer can also be detected on the basis of thealteration of wild-type MMSC2 polypeptide. Such alterations can bedetermined by sequence analysis in accordance with conventionaltechniques. More preferably, antibodies (polyclonal or monoclonal) areused to detect differences in, or the absence of MMSC2 peptides.Techniques for raising and purifying antibodies are well known in theart and any such techniques may be chosen to achieve the preparationsclaimed in this invention. In a preferred embodiment of the invention,antibodies will immunoprecipitate MMSC2 proteins from solution as wellas react with these proteins on Western or immunoblots of polyacrylamidegels. In another preferred embodiment, antibodies will detect MMSC2proteins in paraffin or frozen tissue sections, using immunocytochemicaltechniques.

[0159] Preferred embodiments relating to methods for detecting MMSC2 orits mutations include enzyme linked immunosorbent assays (ELISA),radioimmunoassays (RIA), immunoradiometric assays (IA) andimmunoenzymatic assays (IEMA), including sandwich assays usingmonoclonal and/or polyclonal antibodies. Exemplary sandwich assays aredescribed by David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530,hereby incorporated by reference.

[0160] Alternatively, alterations in the MMSC2 sequence can bedetermined by detecting alterations in the interaction of MMSC2 withMMAC1 or the C-terminus of MMAC1. Wild-type MMAC1 or its C-terminus canbe bound to a solid phase and the interaction with MMSC2 assayed byconventional techniques. Analogously, alterations in MMAC1 which affectits interaction with MMSC2 can be detected using wild-type MMSC2 or itsPDZ domain which interacts with MMAC1 bound to a solid phase.

[0161] Methods of Use: Rational Drug Design

[0162] The goal of rational drug design is to produce structural analogsof biologically active polypeptides of interest or of small moleculeswith which they interact (e.g., agonists, antagonists, inhibitors) inorder to fashion drugs which are, for example, more active or stableforms of the polypeptide, or which, e.g., enhance or interfere with thefunction of a polypeptide in vivo. See, e.g., Hodgson, 1991. In oneapproach, one first determines the three-dimensional structure of aprotein of interest (e.g., MMSC2 polypeptide) by x-ray crystallography,by computer modeling or most typically, by a combination of approaches.Less often, useful information regarding the structure of a polypeptidemay be gained by modeling based on the structure of homologous proteins.An example of rational drug design is the development of HIV proteaseinhibitors (Erickson et al., 1990). In addition, peptides (e.g., MMSC2polypeptide) are analyzed by an alanine scan (Wells, 1991). In thistechnique, an amino acid residue is replaced by Ala, and its effect onthe peptide's activity is determined. Each of the amino acid residues ofthe peptide is analyzed in this manner to determine the importantregions of the peptide.

[0163] It is also possible to isolate a target-specific antibody,selected by a functional assay, and then to solve its crystal structure.In principle, this approach yields a pharmacore upon which subsequentdrug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced banks of peptides. Selected peptides would thenact as the pharmacore.

[0164] Thus, one may design drugs which have, e.g., improved MMSC2polypeptide activity or stability or which act as inhibitors, agonists,antagonists, etc. of MMSC2 polypeptide activity. By virtue of theavailability of cloned MMSC2 sequence, sufficient amounts of the MMSC2polypeptide may be made available to perform such analytical studies asx-ray crystallography. In addition, the knowledge of the MMSC2 proteinsequence provided herein will guide those employing computer modelingtechniques in place of, or in addition to x-ray crystallography.

[0165] Methods of Use: Gene Therapy

[0166] According to the present invention, a method is also provided ofsupplying wild-type MMSC2 function to a cell which carries a mutantMMSC2 allele. Supplying such a function should allow normal functioningof the recipient cells. The wild-type gene or a part of the gene may beintroduced into the cell in a vector such that the gene remainsextrachromosomal. In such a situation, the gene will be expressed by thecell from the extrachromosomal location. More preferred is the situationwhere the wild-type gene or a part thereof is introduced into the mutantcell in such a way that it recombines with the endogenous mutant genepresent in the cell. Such recombination requires a double recombinationevent which results in the correction of the gene mutation. Vectors forintroduction of genes both for recombination and for extrachromosomalmaintenance are known in the art, and any suitable vector may be used.Methods for introducing DNA into cells such as electroporation, calciumphosphate co-precipitation and viral transduction are known in the art,and the choice of method is within the competence of the practitioner.

[0167] As generally discussed above, the MMSC2 gene or fragment, whereapplicable, may be employed in gene therapy methods in order to increasethe amount of the expression products of such gene in cells. It may alsobe useful to increase the level of expression of the MMSC2 gene even inthose persons in which the mutant gene is expressed at a “normal” level,but the gene product is not fully functional.

[0168] Gene therapy would be carried out according to generally acceptedmethods, for example, as described by Friedman (1991) or Culver (1996).Cells from a patient would be first analyzed by the diagnostic methodsdescribed above, to ascertain the production of MMSC2 polypeptide in thecells. A virus or plasmid vector (see further details below), containinga copy of the MMSC2 gene linked to expression control elements, isprepared. The vector may be capable of replicating inside the cells.Alternatively, the vector may be replication deficient and is replicatedin helper cells for use in gene therapy. Suitable vectors are known,such as disclosed in U.S. Pat. No. 5,252,479, published PCT applicationWO 93/07282 and U.S. Pat. No. 5,691,198. The vector is then injectedinto the patient. If the transfected gene is not permanentlyincorporated into the genome of each of the targeted cells, thetreatment may have to be repeated periodically.

[0169] Gene transfer systems known in the art may be useful in thepractice of the gene therapy methods of the present invention. Theseinclude viral and nonviral transfer methods. A number of viruses havebeen used as gene transfer vectors or as the basis for preparing genetransfer vectors, including papovaviruses (e.g., SV40, Madzak et al.,1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia andKapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinsonet al., 1992; Stratford-Perricaudet et al., 1990; Schneider et al.,1998), vaccinia virus (Moss, 1992), adeno-associated virus (Muzyczka,1992; Ohi et al., 1990; Russell & Hirata, 1998), herpesviruses includingHSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink et al., 1992;Breakfield and Geller, 1987; Freese et al., 1990; Fink et al., 1996),lentiviruses (Naldini et al., 1996), vaccinia virus (Moss, 1996),Sindbis and Semliki Forest virus (Berglund et al., 1993), andretroviruses of avian (Brandyopadhyay and Temin, 1984; Petropoulos etal., 1992), murine (Miller, 1992; Miller et al., 1985; Sorge et al.,1984; Mann and Baltimore, 1985; Miller et al., 1988), and human origin(Shimada et al., 1991; Helseth et al., 1990; Page et al., 1990;Buchschacher and Panganiban, 1992). Most human gene therapy protocolshave been based on disabled murine retroviruses, although adenovirus andadeno-associated virus are also being used.

[0170] Nonviral gene transfer methods known in the art include chemicaltechniques such as calcium phosphate coprecipitation (Graham and van derEb, 1973; Pellicer et al., 1980); mechanical techniques, for examplemicroinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster etal., 1981; Constantini and Lacy, 1981); membrane fusion-mediatedtransfer via liposomes (Felgner et al., 1987; Wang and Huang, 1989;Kaneda et al., 1989; Stewart et al., 1992; Nabel et al., 1990; Lim etal., 1992); and direct DNA uptake and receptor-mediated DNA transfer(Wolff et al., 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al.,1989b; Wolff et al., 1991; Wagner et al., 1990; Wagner et al., 1991;Cotten et al., 1990; Curiel et al., 1991a; Curiel et al., 1991b).

[0171] In an approach which combines biological and physical genetransfer methods, plasmid DNA of any size is combined with apolylysine-conjugated antibody specific to the adenovirus hexon protein,and the resulting complex is bound to an adenovirus vector. Thetrimolecular complex is then used to infect cells. The adenovirus vectorpermits efficient binding, internalization, and degradation of theendosome before the coupled DNA is damaged. For other techniques for thedelivery of adenovirus based vectors Schneider et al. (1998) and U.S.Pat. No. 5,691,198.

[0172] Liposome/DNA complexes have been shown to be capable of mediatingdirect in vivo gene transfer. While in standard liposome preparationsthe gene transfer process is nonspecific, localized in vivo uptake andexpression have been reported in tumor deposits, for example, followingdirect in situ administration (Nabel, 1992).

[0173] Expression vectors in the context of gene therapy are meant toinclude those constructs containing sequences sufficient to express apolynucleotide that has been cloned therein. In viral expressionvectors, the construct contains viral sequences sufficient to supportpackaging of the construct. If the polynucleotide encodes MMSC2,expression will produce MMSC2. If the polynucleotide encodes anantisense polynucleotide or a ribozyme, expression will produce theantisense polynucleotide or ribozyme. Thus in this context, expressiondoes not require that a protein product be synthesized. In addition tothe polynucleotide cloned into the expression vector, the vector alsocontains a promoter functional in eukaryotic cells. The clonedpolynucleotide sequence is under control of this promoter. Suitableeukayotic promoters include those described above. The expression vectormay also include sequences, such as selectable markers and othersequences described herein.

[0174] Gene transfer techniques which target DNA directly to braintissue is preferred. Receptor-mediated gene transfer, for example, isaccomplished by the conjugation of DNA (usually in the form ofcovalently closed supercoiled plasmid) to a protein ligand viapolylysine. Ligands are chosen on the basis of the presence of thecorresponding ligand receptors on the cell surface of the targetcell/tissue type. These ligand-DNA conjugates can be injected directlyinto the blood if desired and are directed to the target tissue wherereceptor binding and internalization of the DNA-protein complex occurs.To overcome the problem of intracellular destruction of DNA, coinfectionwith adenovirus can be included to disrupt endosome function.

[0175] The therapy is as follows: patients who carry a MMSC2susceptibility allele are treated with a gene delivery vehicle such thatsome or all of their brain precursor cells receive at least oneadditional copy of a functional normal MMSC2 allele, respectively. Inthis step, the treated individuals have reduced risk of cancer to theextent that the effect of the susceptible allele has been countered bythe presence of the normal allele.

[0176] Methods of Use: Peptide Therapy

[0177] Peptides which have MMSC2 activity can be supplied to cells whichcarry a mutant or missing MMSC2 allele. Protein can be produced byexpression of the cDNA sequence in bacteria, for example, using knownexpression vectors. Alternatively, MMSC2 polypeptide can be extractedfrom MMSC2-producing mammalian cells. In addition, the techniques ofsynthetic chemistry can be employed to synthesize MMSC2 protein. Any ofsuch techniques can provide the preparation of the present inventionwhich comprises the MMSC2 protein. The preparation is substantially freeof other human proteins. This is most readily accomplished by synthesisin a microorganism or in vitro.

[0178] Active MMSC2 molecules can be introduced into cells bymicroinjection or by use of liposomes, for example. Alternatively, someactive molecules may be taken up by cells, actively or by diffusion.Supply of molecules with MMSC2 activity should lead to inhibition ofcancer. Other molecules with MMSC2 activity (for example, peptides,drugs or organic compounds) may also be used to effect such aninhibition. Modified polypeptides having substantially similar functionare also used for peptide therapy.

[0179] Methods of Use: Transformed Hosts

[0180] Animals for testing therapeutic agents can be selected aftermutagenesis of whole animals or after treatment of germline cells orzygotes. Such treatments include insertion of mutant MMSC2 alleles,usually from a second animal species, as well as insertion of disruptedhomologous genes. Alternatively, the endogenous MMSC2 gene of theanimals may be disrupted by insertion or deletion mutation or othergenetic alterations using conventional techniques (Capecchi, 1989;Valancius and Smithies, 1991; Hasty et al., 1991; Shinkai et al., 1992;Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992;Donehower et al., 1992). After test substances have been administered tothe animals, the presence of cancer must be assessed. If the testsubstance prevents or suppresses the appearance of cancer, then the testsubstance is a candidate therapeutic agent for treatment of cancer.These animal models provide an extremely important testing vehicle forpotential therapeutic products.

[0181] Methods of Use: Transgenic/Knockout Animals and Models

[0182] In one embodiment of the invention, transgenic animals areproduced which contain a functional transgene encoding a functionalMMSC2 polypeptide or variants thereof. Transgenic animals expressingMMSC2 transgenes, recombinant cell lines derived from such animals andtransgenic embryos may be useful in methods for screening for andidentifying agents that induce or repress function of MMSC2. Transgenicanimals of the present invention also can be used as models for studyingindications such as cancers.

[0183] In one embodiment of the invention, a MMSC2 transgene isintroduced into a non-human host to produce a transgenic animalexpressing a human or murine MMSC2 gene. The transgenic animal isproduced by the integration of the transgene into the genome in a mannerthat permits the expression of the transgene. Methods for producingtransgenic animals are generally described by Wagner and Hoppe (U.S.Pat. No. 4,873,191; which is incorporated herein by reference), Brinsteret al. 1985; which is incorporated herein by reference in its entirety)and in “Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition(eds., Hogan, Beddington, Costantimi and Long, Cold Spring HarborLaboratory Press, 1994; which is incorporated herein by reference in itsentirety).

[0184] It may be desirable to replace the endogenous MMSC2 by homologousrecombination between the transgene and the endogenous gene; or theendogenous gene may be eliminated by deletion as in the preparation of“knock-out” animals. Typically, a MMSC2 gene flanked by genomicsequences is transferred by microinjection into a fertilized egg. Themicroinjected eggs are implanted into a host female, and the progeny arescreened for the expression of the transgene. Transgenic animals may beproduced from the fertilized eggs from a number of animals including,but not limited to reptiles, amphibians, birds, mammals, and fish.Within a particularly preferred embodiment, transgenic mice aregenerated which overexpress MMSC2 or express a mutant form of thepolypeptide. Alternatively, the absence of a MMSC2 in “knock-out” micepermits the study of the effects that loss of MMSC2 protein has on acell in vivo. Knock-out mice also provide a model for the development ofMMSC2-related cancers.

[0185] Methods for producing knockout animals are generally described byShastry (1995, 1998) and Osterrieder and Wolf (1998). The production ofconditional knockout animals, in which the gene is active until knockedout at the desired time is generally described by Feil et al. (1996),Gagneten et al. (1997) and Lobe and Nagy (1998). Each of thesereferences is incorporated herein by reference.

[0186] As noted above, transgenic animals and cell lines derived fromsuch animals may find use in certain testing experiments. In thisregard, transgenic animals and cell lines capable of expressingwild-type or mutant MMSC2 may be exposed to test substances. These testsubstances can be screened for the ability to enhance wild-type MMSC2expression and or function or impair the expression or function ofmutant MMSC2.

[0187] Pharmaceutical Compositions and Routes of Administration

[0188] The MMSC2 polypeptides, antibodies, peptides and nucleic acids ofthe present invention can be formulated in pharmaceutical compositions,which are prepared according to conventional pharmaceutical compoundingtechniques. See, for example, Remington's Pharmaceutical Sciences, 18thEd. (1990, Mack Publishing Co., Easton, Pa.). The composition maycontain the active agent or pharmaceutically acceptable salts of theactive agent. These compositions may comprise, in addition to one of theactive substances, a pharmaceutically acceptable excipient, carrier,buffer, stabilizer or other materials well known in the art. Suchmaterials should be non-toxic and should not interfere with the efficacyof the active ingredient. The carrier may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.,intravenous, oral, intrathecal, epineural or parenteral.

[0189] For oral administration, the compounds can be formulated intosolid or liquid preparations such as capsules, pills, tablets, lozenges,melts, powders, suspensions or emulsions. In preparing the compositionsin oral dosage form, any of the usual pharmaceutical media may beemployed, such as, for example, water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents, suspending agents, andthe like in the case of oral liquid preparations (such as, for example,suspensions, elixirs and solutions); or carriers such as starches,sugars, diluents, granulating agents, lubricants, binders,disintegrating agents and the like in the case of oral solidpreparations (such as, for example, powders, capsules and tablets).Because of their ease in administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe sugar-coated or enteric-coated by standard techniques. The activeagent can be encapsulated to make it stable to passage through thegastrointestinal tract while at the same time allowing for passageacross the blood brain barrier. See for example, WO 96/11698.

[0190] For parenteral administration, the compound may dissolved in apharmaceutical carrier and administered as either a solution of asuspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative or synthetic origin. The carrier may also contain otheringredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administeredintrathecally, they may also be dissolved in cerebrospinal fluid.

[0191] The active agent is preferably administered in an therapeuticallyeffective amount. The actual amount administered, and the rate andtime-course of administration, will depend on the nature and severity ofthe condition being treated. Prescription of treatment, e.g. decisionson dosage, timing, etc., is within the responsibility of generalpractitioners or spealists, and typically takes account of the disorderto be treated, the condition of the individual patient, the site ofdelivery, the method of administration and other factors known topractitioners. Examples of techniques and protocols can be found inRemington's Parmaceutical Sciences.

[0192] Alternatively, targeting therapies may be used to deliver theactive agent more specifically to certain types of cell, by the use oftargeting systems such as antibodies or cell specific ligands. Targetingmay be desirable for a variety of reasons, e.g. if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.

[0193] Instead of administering these agents directly, they could beproduced in the target cell, e.g. in a viral vector such as describedabove or in a cell based delivery system such as described in U.S. Pat.No. 5,550,050 and published PCT application Nos. WO 92/19195, WO94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO96/40871, WO 96/40959 and WO 97/12635. designed for implantation in apatient. The vector could be targeted to the specific cells to betreated, or it could contain regulatory elements which are more tissuespecific to the target cells. The cell based delivery system is designedto be implanted in a patient's body at the desired target site andcontains a coding sequence for the active agent. Alternatively, theagent could be administered in a precursor form for conversion to theactive form by an activating agent produced in, or targeted to, thecells to be treated. See for example, EP 425,731A and WO 90/07936.

[0194] The identification of the association between the MMSC2 genemutations and cancer permits the early presymptomatic screening ofindividuals to identify those at risk for developing cancer. To identifysuch individuals, MMSC2 alleles are screened for mutations eitherdirectly or after cloning the alleles. The alleles are tested for thepresence of nucleic acid sequence differences from the normal alleleusing any suitable technique, including but not limited to, one of thefollowing methods: fluorescent in situ hybridization (FISH), direct DNAsequencing, PFGE analysis, Southern blot analysis, single strandedconformation analysis (SSCP), linkage analysis, RNase protection assay,allele specific oligonucleotide (ASO), dot blot analysis and PCR-SSCPanalysis. Also useful is the recently developed technique of DNAmicrochip technology. For example, either (1) the nucleotide sequence ofboth the cloned alleles and normal MMSC2 gene or appropriate fragment(coding sequence or genomic sequence) are determined and then compared,or (2) the RNA transcripts of the MMSC2 gene or gene fragment arehybridized to single stranded whole genomic DNA from an individual to betested, and the resulting heteroduplex is treated with Ribonuclease A(RNase A) and run on a denaturing gel to detect the location of anymismatches. Two of these methods can be carried out according to thefollowing procedures.

[0195] The alleles of the MMSC2 gene in an individual to be tested arecloned using conventional techniques. For example, a blood sample isobtained from the individual. The genomic DNA isolated from the cells inthis sample is partially digested to an average fragment size ofapproximately 20 kb. Fragments in the range from 18-21 kb are isolated.The resulting fragments are ligated into an appropriate vector. Thesequences of the clones are then determined and compared to the normalMMSC2 gene.

[0196] Alternatively, polymerase chain reactions (PCRs) are performedwith primer pairs for the 5′ region or the exons of the MMSC2 gene. PCRscan also be performed with primer pairs based on any sequence of thenormal MMSC2 gene. For example, primer pairs for one of the introns canbe prepared and utilized. Finally, RT-PCR can also be performed on themRNA. The amplified products are then analyzed by single strandedconformation polymorphisms (SSCP) using conventional techniques toidentify any differences and these are then sequenced and compared tothe normal gene sequence.

[0197] Individuals can be quickly screened for common MMSC2 genevariants by amplifying the individual's DNA using suitable primer pairsand analyzing the amplified product, e.g., by dot-blot hybridizationusing allele-specific oligonucleotide probes.

[0198] The second method employs RNase A to assist in the detection ofdifferences between the normal MMSC2 gene and defective genes. Thiscomparison is performed in steps using small (˜500 bp) restrictionfragments of the MMSC2 gene as the probe. First, the MMSC2 gene isdigested with a restriction enzyme(s) that cuts the gene sequence intofragments of approximately 500 bp. These fragments are separated on anelectrophoresis gel, purified from the gel and cloned individually, inboth orientations, into an SP6 vector (e.g., pSP64 or pSP65). TheSP6-based plasmids containing inserts of the MMSC2 gene fragments aretranscribed in vitro using the SP6 transcription system, well known inthe art, in the presence of [α-³²P]GTP, generating radiolabeled RNAtranscripts of both strands of the gene.

[0199] Individually, these RNA transcripts are used to formheteroduplexes with the allelic DNA using conventional techniques.Mismatches that occur in the RNA:DNA heteroduplex, owing to sequencedifferences between the MMSC2 fragment and the MMSC2 allele subclonefrom the individual, result in cleavage in the RNA strand when treatedwith RNase A. Such mismatches can be the result of point mutations orsmall deletions in the individual's allele. Cleavage of the RNA strandyields two or more small RNA fragments, which run faster on thedenaturing gel than the RNA probe itself.

[0200] Any differences which are found, will identify an individual ashaving a molecular variant of the MMSC2 gene and the consequent presenceof cancer. These variants can take a number of forms. The most severeforms would be frame shift mutations or large deletions which wouldcause the gene to code for an abnormal protein or one which wouldsignificantly alter protein expression. Less severe disruptive mutationswould include small in-frame deletions and nonconservative base pairsubstitutions which would have a significant effect on the proteinproduced, such as changes to or from a cysteine residue, from a basic toan acidic amino acid or vice versa, from a hydrophobic to hydrophilicamino acid or vice versa, or other mutations which would affectsecondary or tertiary protein structure. Silent mutations or thoseresulting in conservative amino acid substitutions would not generallybe expected to disrupt protein function.

[0201] Genetic testing will enable practitioners to identify individualsat risk for cancer at, or even before, birth. Finally, this inventionchanges our understanding of the cause and treatment of cancer.

EXAMPLES

[0202] The present invention is further detailed in the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below are utilized.

Example 1 Identification of MMSC2

[0203] A yeast two-hybrid assay was performed using conventionaltechniques, such as described by Fields and Song (1989), Chevray andNathans (1992), Bartel et al. (1993) and Lee et al. (1995). Sequenceencoding the C-terminal 15 amino acids of MMAC1 (NEPFDEDQHTQITKV; SEQ IDNO: 4) plus its stop codon was generated using an oligonucleotidesynthesizer and was ligated to plasmid pGBT.C such that the codingsequence of MMAC1 was in-frame with coding sequence for the Ga14pDNA-binding domain. This plasmid construct was introduced into the yeastreporter strain J692 along with a library of activation domain fusionplasmids prepared from human kidney cDNA (Clontech). Transformants werespread onto 20-150 mm plates of yeast minimal media lacking leucine,tryptophan, and histidine, and containing 25 mM 3-amino-1,2,4-triazole(Gietz et al., 1995; Bartel and Fields, 1995). After one week incubationat 30° C., yeast colonies were assayed for expression of the lacZreporter gene by beta-galactosidase filter assay (Breeden and Naysmyth,1985). Colonies that both grew in the absence of histidine and werepositive for production of beta-galactosidase were chosen for furthercharacterization.

[0204] The activation domain plasmid was purified from positive coloniesby the smash-and-grab technique. These plasmids were introduced into E.coli DH10B (Gibco BRL) by electroporation and purified from E. coli bythe alkaline lysis method. To test for the specificity of theinteraction, specific activation domain plasmids were cotransformed intostrain J692 with plasmids encoding various DNA-binding domain fusionproteins, including fusions to C-terminal segments of MMAC1 and humanlamin C. Transformants from these experiments were assayed forexpression of the HIS3 and lacZ reporter genes. Positives that expressedreporter genes with MMAC1 constructs and not with lamin C constructsencode bona fide MMAC1-interacting proteins. These proteins wereidentified and characterized by sequence analysis of the insert of theappropriate activation domain plasmid.

[0205] Five of the clones encoding bona fide MMAC1-interacting proteinswere named PDZBN2B, PDZBN3A, PDZBN5B, PDZBN18D, and pdzk4. Comparison ofthe sequences of these clones suggested that they were all partial cDNAsderived from the same novel gene. A search of GenBank with thesesequences revealed strong sequence similarity with a partial mouse cDNAsequence called 9ORF binding protein 1 (9BP-1)(GenBank Accession #AF000168).

[0206] Several rounds of cDNA library screening were required toidentify cDNA clones that could be assembled into the full length MMSC2sequence. In the first round, a 509 base pair(bp) probe was developedfrom the 5′ end of clone PDZBN2B using primers

[0207] 9BP-1 F1: AGACAGCAAAGATGACAGTAA (SEQ ID NO: 5) and

[0208] 9BP-1 R4: CTTCCTCCTCTTTGTATGGG (SEQ ID NO: 6).

[0209] This probe was used to screen a human placental cDNA library anda human prostate cDNA library. Two of the informative clones obtainedwere p18a (placental) and pr63(prostate). A search of GenBank with thisadditional sequence yielded an additional human EST (GenBank Accesion #C75629). For the second round of cDNA library screening, a 202 bp probewas developed from the 5′ end of this EST using primers

[0210] 9BP-1 #1: GCTTTTGCCGAAATGGGTAGT (SEQ ID NO: 7) and

[0211] 9BP-1 #2: GATCGGTCTTTGTTCCCAGCA (SEQ ID NO: 8).

[0212] This probe was used to screen a human prostate cDNA library; twoof the informative clones obtained were clone #10 and clone #3. For thethird round of cDNA library screening, a 172 bp probe was developed fromthe 5′ end of clone #3 using primers

[0213] 9BP-1 #5: TGTGAGCAAGTTTAGTGAG (SEQ ID NO: 9) and

[0214] 9BP-1 #7: GGTGATTTTCCCCAAGTAA (SEQ ID NO: 10)

[0215] and used to screen a human prostate cDNA library. One of theresulting clones, clone # 6, yielded the start codon and part of the 5′UTR, including in-frame upstream stop codons. The nucleotide sequencefor MMSC2 is set forth in SEQ ID NO: 2 with the amino acid sequence ofthe encoded protein set forth in SEQ ID NO: 3.

[0216]FIG. 1 shows a diagram of MMSC2 indicating the position of ORF andthe positions of the 13 PDZ domains. FIG. 2 shows a diagram of the keyclones used to assemble the full length MMSC2 sequence, the probes usedto identify those clones, and the relative position of the partiallysequenced mouse ortholog 9BP-1(Accession # AF000168). Within the 5′ UTR,the in-frame upstream stop codon at nucleotide 42 demonstrates that thestart codon at nucleotide 57 has been correctly identified. The 13 PDZdomains correspond to the amino acids of MMSC2 as shown in Table 1.TABLE 1 Sequence Correspondence of 13 PDZ Domains Domain Number DomainName Amino Acid Span 1 P15 136-222 2 P14 256-335 3 P13 376-461 4 P10555-632 5 P12 699-785 6 P11 1007-1090 7 P9 1150-1241 8 P8 1316-1398 9 P71439-1529 10  P1 1595-1677 11  P2 1691-1772 12  P3 1828-1913 13  P41953-2037

Example 2 Identification of MMSC2-interacting Proteins by two-hybridAnalysis

[0217] DNA fragments encoding all or portions of MMSC2 are ligated to atwo-hybrid DNA-binding domain vector such as pGBT.C such that the codingsequence of MMSC2 is in-frame with coding sequence for the Ga14pDNA-binding domain. These DNA fragments may encode specific PDZ domainsof MMSC2 plus the 5 to 10 amino acids N- and C-terminal of each specificPDZ. A plasmid that encodes a DNA-binding domain fusion to a fragment ofMMSC2 PDZ is introduced into the yeast reporter strain (such as J692)along with a library of cDNAs fused to an activation domain.Transformants are spread onto 20-150 mm plates of selective media, suchas yeast minimal media lacking leucine, tryptophan, and histidine, andcontaining 25 mM 3-amino-1,2,4-triazole. After one week incubation at30° C., yeast colonies are assayed for expression of the lacZ reportergene by beta-galactosidase filter assay. Colonies that both grow in theabsence of histidine and are positive for production ofbeta-galactosidase are chosen for further characterization.

[0218] The activation domain plasmid is purified from positive coloniesby the smash-and-grab technique. These plasmids are introduced into E.coli (e.g., DH10B (Gibco BRL)) by electroporation and purified from E.coli by the alkaline lysis method. To test for the specificity of theinteraction, specific activation domain plasmids are cotransformed intostrain J692 with plasmids encoding various DNA-binding domain fusionproteins, including fusions to segments of MMSC2 and human lamin C.Transformants from these experiments are assayed for expression of theHIS3 and lacZ reporter genes. Positives that express reporter genes withMMSC2 constructs and not with lamin C constructs encode bona fideMMSC2-interacting proteins. These proteins are identified andcharacterized by sequence analysis of the insert of the appropriateactivation domain plasmid.

Example 3 Characterization of the Binding Specificity of MMSC2 PDZDomains by Two-hybrid Analysis

[0219] DNA fragments encoding specific PDZ domains of MMSC2 plus the 5to 10 amino acids N- and C-terminal of each specific PDZ domain aregenerated by PCR amplification. These fragments are ligated to atwo-hybrid DNA-binding domain vector such as pGBT.C such that the codingsequence of MMSC2 is in-frame with coding sequence for the Ga14pDNA-binding domain. An activation domain library is prepared thatencodes an activation domain fused in-frame to random peptide sequencesthat end with a stop codon. An example of this type of library is theClontech random peptide library. A plasmid that encodes a DNA-bindingdomain fusion to a specific MMSC2 PDZ domain is introduced into theyeast reporter strain (such as J692) along with a library of randompeptides fused to an activation domain. Transformants are spread onto20-150 mm plates of selective media, such as yeast minimal media lackingleucine, tryptophan, and histidine, and containing 25 mM3-amino-1,2,4-triazole. After one week incubation at 30° C., yeastcolonies are assayed for expression of the lacZ reporter gene bybeta-galactosidase filter assay. Colonies that both grow in the absenceof histidine and are positive for production of beta-galactosidase arechosen for sequence analysis. The insert of the activation domainconstruct is characterized by sequence analysis. The sequence of thepeptide that binds to the MMSC2 PDZ domain is obtained by conceptualtranslation of the nucleotide sequence. Peptide sequences from multipleisolates are aligned to determine a consensus binding motif. This motifcan be used to identify cellular proteins that bind MMSC2 and to developsmall molecules that inhibit binding to these specific PDZ domains.

Example 4 In vitro Protein-protein Interaction Assay

[0220] cDNAs encoding each of the MMSC2 PDZ domains (amino acid residuesidentified in Table 1), and any desired control proteins, were generatedby PCR and subcloned as glutathione S-transferase (GST) fusions in pGEXvectors (Pharmacia). After sequencing to confirm expression constructintegrity, the resulting clones were expressed in E. coli and thedesired fusion proteins isolated with glutathione-agarose and recoveredwith glutathione elution. These fusion proteins or control proteins werethen adsorbed to different wells of a 96-well ELISA plate and remainingsites blocked with BSA. Synthetic commercially synthesized peptidesencoding the desired PDZ-binding domain (i.e., the 16 C-terminal aminoacids of MMAC1, or the C-terminal peptide sequences of interactingproteins identified by the approach of Example 2, or the C-terminalpeptide sequences identified by the approach of Example 3), or a controlpeptide, and biotinylated at the amino-terminus, were pre-bound tostreptavidin-alkaline phosphatase in a 4:1 molar ratio. The biotinylatedpeptide streptavidin-alkaline phosphatase complexes were then blockedwith free biotin. These pre-bound peptide streptavidin-alkalinephosphatase complexes were then incubated with the immobilized PDZdomains in wash buffer containing PBS, BSA and triton-X100. Unboundmaterial was removed with repeated washes. Boundpeptide/streptavidin-alkaline phosphatase complex was then quantitatedby a colorimetric phosphatase assay read on a 96-well plate reader. Thefollowing peptides were used in the initial study:

[0221] SH3 binding peptide: biotin-SGSGILAPPVPPRNTR-COOH (SEQ ID NO: 11)

[0222] MMAC1.388-403: biotin-ENEPFDEDQHTQITKV-COOH (SEQ ID NO: 12).

[0223] The relative affinity of each of the 13 PDZ domains encoded byMMSC2 for an MMAC1 C-terminal PDZ peptide as measured by an ELISA assayis set forth in Table 2. TABLE 2 PDZ Binding Assay Domain Number DomainName Peptide A405 1 P15 MMAC1 0.011 SH3 0.007 2 P14 MMAC1 0.001 SH30.006 3 P13 MMAC1 0.006 SH3 0.008 4 P10 MMAC1 0.024 SH3 0.010 5 P12MMAC1 0.000 SH3 0.005 6 P11 MMAC1 0.016 SH3 0.017 7 P9 MMAC1 0.667 SH30.006 8 P8 MMAC1 0.006 SH3 0.008 9 P7 MMAC1 0.017 SH3 0.009 10  P1 MMAC10.174 SH3 0.002 11  P2 MMAC1 0.012 SH3 0.009 12  P3 MMAC1 0.003 SH30.011 13  P4 MMAC1 0.456 SH3 0.006

[0224] The GST-affinity pull down assay is a complementary in vitromethod for investigating protein-protein interactions. PDZ domain-GSTfusion proteins are incubated with synthetic biotinylated peptides inwash buffer (these peptides were described above). Streptavidin magneticbeads are then added to recover the biotinylated peptide, then unboundmaterial removed by washing. The beads are then incubated with SDS/DTTloading buffer at 100° C. and bound protein detected by SDS/PAGE andcoomasie blue staining.

Example 5 Mutation Screening of MMSC2

[0225] Nested PCR amplifications were performed on cDNA from tumor celllines. Total cell line RNAs were reverse transcribed with Superscript II(Life Technologies) and random hexamers. Using the outer primer pairfrom each amplicon (i.e. 9BP.1A and 9BP.1P or 9BP.2A and 9BP.2P),approximately 10 ng of cDNA from each cell line was amplified for 26cycles. Products were diluted 60 fold and then reamplified for 22-26cycles using nested M13 tailed primers (i.e. 9BP.1B and 9BP.1Q or 9BP.2Band 9BP.2Q). Typical primary amplification cycling conditions were aninitial denaturation at 95° C. for 60s, followed by 26 cycles of 96° C.(12s), 58° C. (15s) and 72° C. (90s). Typical secondary amplificationcycling conditions were an initial denaturation at 95° C. for 60s,followed by 22-26 cycles of 96° C. (12s), 58° C. (15s) and 72° C. (40s).The resulting RT-PCR products were sequenced with dye-primer chemistryon ABI 377 sequencers. Sequences were examined for the presence ofvariants using the program Sequencher.

[0226] The primers used are set forth in Table 3. The sequence variantsare set forth in Table 4. TABLE 3 Table of Primers Primer SEQ IDSequence 9BP.1A 13 GCCACCGCGGGATTAAGTTTCT 9BP.1P 14TGTAGCCAGCAATGGTAATTCCT 9BP.1B 15GTTTTCCCAGTCACGACGGTTCCATTTTAATTGCTGTTAAT 9BP.1Q 16AGGAAACAGCTATGACCATGGGGATAATAAAAACGATTCATTT 9BP.1C 17GTTTTCCCAGTCACGACGTTGAATATGCCCACGTTCCTC 9BP.1R 18AGGAAACAGCTATGACCATTCTTTCAATCTTCCATCTCTATG 9BP.1D 19GTTTTCCCAGTCACGACGAACAGAGGAGAGCTGGGAATA 9BP.1S 20AGGAAACAGCTATGACCATCAAACCAGATCCATCATTCACC 9BP.1E 21GTTTTCCCAGTCACGACGGCACAATTTCAGCTCACTCTAA 9BP.1T 22AGGAAACAGCTATGACCATGGATGAGGAGAGGGTGATGC 9BP.2A 23 TCTAGCAGCAATGAGCAGTGAG9BP.2P 24 GATCCTGATAATCTAAAATGCTAA 9BP.2B 25GTTTTCCCAGTCACGACGAAGTTGATGATTGCAAGAGGTG 9BP.2Q 26AGGAAACAGCTATGACCATGGTTTGTGCCATCTACTGCTAT 9BP.2C 27GTTTTCCCAGTCACGACAGAAAGCAGTGCCGTTGAGCATG 9BP.2R 28AGGAAACAGCTATGACCATGCTGACAGTAATGGATACCCT 9BP.2D 29GTTTTCCCAGTCACGACGGATTTTTTATCTTCGAGAGAAA 9BP.2S 30AGGAAACAGCTATGACCATTTCCCCAAGTAAAGTTATGCCAT 9BP.2E 31GTTTTCCCAGTCACGACGTCCTGTTGGACACAGCGGGA 9BP.2T 32AGGAAACAGCTATGACCATCATGGCCAAAGGTGCTTGAA 9BP.3A 33 CCACCCACCACCCAATCAGAAT9BP.3P 34 CATCTCGACTAATGGCACCTCC 9BP.3B 35GTTTTCCCAGTCACGACGGAGACAGAGGATCCAGTGCT 9BP.3Q 36AGGAAACAGCTATGACCATCCCTGACGGTGCTCCCTTCA 9BP.3C 37GTTTTCCCAGTCACGACGTTAACTTGGAAAACAGCAGTCT 9BP.3R 38AGGAAACAGCTATGACCATCATCACCACAAGAACTGCCATG 9BP.3D 39GTTTTCCCAGTCACGACGACTCTCCTGAAAATGACAGCAT 9BP.3S 40AGGAAACAGCTATGACCATTAAATGAGATTCAGTCCACACT 9BP.3E 41GTTTTCCCAGTCACGACGATAAATGACTACACACCTGCAA 9BP.3T 42AGGAAACAGCTATGACCATAACGATCATCCCCAAGCCATCT 9BP.4A 43CTGAGTACCTGCTTGAACAGAG 9BP.4P 44 GACCATTGATCTCTAGAAGCTC 9BP.4B 45GTTTTCCCAGTCACGACGGGACTATTAATATAGCAAAAGGC 9BP.4Q 46AGGAAACAGCTATGACCATCAGTGCCATTACTCTTCCAGA 9BP.4C 47GTTTTCCCAGTCACGACGTACTTATGTGCCTGCAGAACA 9BP.4R 48AGGAAACAGCTATGACCATCATGTTTGATGAAAATGCCCC 9BP.4D 49GTTTTCCCAGTCACGACGATTGTTGGTGGACGAGGGATG 9BP.4S 50AGGAAACAGCTATGACCATCCATTTCGGCAAAGGCTGAAG 9BP.4E 51GTTTTCCCAGTCACGACGCAGAGTCAGAGCCAGAGAAGG 9BP.4T 52AGGAAACAGCTATGACCATAGAAGCTCATCTGCAATTTGC 9BP.5A 53 CAGGCGAGCTGCATATGATTG9BP.5P 54 CCTCCTTTGACAATGTCTGACAC 9BP.5B 55GTTTTCCCAGTCACGACGGTGTCTTCATAGTGGGGATTGAT 9BP.5Q 56AGGAAACAGCTATGACCATGAAGCTCCAGATGTTGCACAT 9BP.5C 57GTTTTCCCAGTCACGACGAGAGCCAACTGTTACTACTTC 9BP.5R 58AGGAAACAGCTATGACCATTGAAGGAACAGCCTGGGAATC 9BP.5D 59GTTTTCCCAGTCACGACGTTAGCCTTCTGAAGACAGCAA 9BP.5S 60AGGAAACAGCTATGACCATCATGGATAATAATGGCACCCA 9BP.5E 61GTTTTCCCAGTCACGACGTTTCCAAAGGGCGAACAGGGC 9BP.5T 62AGGAAACAGCTATGACCATCCAACAATACTTAATCCTAGGC 9BP.6A 63TGGAATTGACTTGAGAAAGGCCA 9BP.6P 64 CCCCCTACAGTTTTGAAGACCC 9BP.6B 65GTTTTCCCAGTCACGACGAAGAGGAGGAAGTGTGTGACAC 9BP.6Q 66AGGAAACAGCTATGACCATGACAGGCTGCCTTCACTCACC 9BP.6C 67GTTTTCCCAGTCACGACGTCAAAGCTGGTCCATTCCATT 9BP.6R 68AGGAAACAGCTATGACCATGGATGTGCCACAGATGGTGAC 9BP.6D 69GTTTTCCCAGTCACGACGATGATGCACCCAACTGGAGTT 9BP.6S 70AGGAAACAGCTATGACCATGGCTGCCATATCCTCCAACTA 9BP.6E 71GTTTTCCCAGTCACGACGGGACCTCCTCAATGTAAGTCT 9BP.6T 72AGGAAACAGCTATGACCATATTGTCAGGACCAGTGCATTC

[0227] Sequence Variants Cell line Type nt variant aa change noteLNCAP.FGC prostatic G163A val->ile heterozygous OV-1063 ovarian G343Tgly->trp non-het* UACC812 breast A1074G thr->thr heterozygous UACC8933breast G5624A arg->lys non-het* HS776T pancreatic G5624A arg->lysnon-het*

Example 6 Generation of Polyclonal Antibody Against MMSC2

[0228] Segments of MMSC2 coding sequence are expressed as fusion proteinin E. coli. The overexpressed protein is purified by gel elution andused to immunize rabbits and mice using a procedure similar to the onedescribed by Harlow and Lane, 1988. This procedure has been shown togenerate Abs against various other proteins (for example, see Kraemer etal., 1993).

[0229] Briefly, a stretch of MMSC2 coding sequence is cloned as a fusionprotein in plasmid PET5A (Novagen, Inc., Madison, Wis.). After inductionwith IPTG, the overexpression of a fusion protein with the expectedmolecular weight is verified by SDS/PAGE. Fusion protein is purifiedfrom the gel by electroelution. Identification of the protein as theMMSC2 fusion product is verified by protein sequencing at theN-terminus. Next, the purified protein is used as immunogen in rabbits.Rabbits are immunized with 100 μg of the protein in complete Freund'sadjuvant and boosted twice in 3 week intervals, first with 100 μg ofimmunogen in incomplete Freund's adjuvant followed by 100 μg ofimmunogen in PBS. Antibody containing serum is collected two weeksthereafter. This procedure is repeated to generate antibodies againstthe mutant forms of the MMSC2 gene product. These antibodies, inconjunction with antibodies to wild type MMSC2, are used to detect thepresence and the relative level of the mutant forms in various tissuesand biological fluids.

Example 7 Generation of Polyclonal Antibody Against MMSC2-MMSC2Interacting Protein Complex

[0230] MMSC2 is capable of binding to certain proteins, e.g., MMAC1. Acomplex of the two proteins is prepared, e.g., by mixing purifiedpreparations of each of the two proteins. If desired, the proteincomplex can be stabilized by cross-linking the proteins in the complexby methods known to those of skill in the art. The protein complex isused to immunize rabbits and mice using a procedure similar to the onedescribed by Harlow and Lane, 1988. This procedure has been shown togenerate Abs against various other proteins (for example, see Kraemer etal., 1993).

[0231] Briefly, the purified protein complex is used as immunogen inrabbits. Rabbits are immunized with 100 μg of the protein in completeFreund's adjuvant and boosted twice in 3 week intervals, first with 100μg of immunogen in incomplete Freund's adjuvant followed by 100 μg ofimmunogen in PBS. Antibody containing serum is collected two weeksthereafter.

[0232] This procedure is repeated to generate antibodies against formsof the complex which comprise mutant MMSC2 or mutant MMSC2 interactingprotein (e.g., mutant MMAC1). These antibodies, in conjunction withantibodies to wild type MMSC2 or MMSC2 interacting protein (e.g.,MMAC1), are used to detect the presence and the relative level of themutant forms in various tissues and biological fluids.

Example 8 Generation of Monoclonal Antibodies Specific for MMSC2

[0233] Monoclonal antibodies are generated according to the followingprotocol. Mice are immunized with immunogen comprising intact MMSC2 orMMSC2 peptides (wild type or mutant) conjugated to keyhole limpethemocyanin using glutaraldehyde or EDC as is well known.

[0234] The immunogen is mixed with an adjuvant. Each mouse receives fourinjections of 10 to 100 μg of immunogen and after the fourth injectionblood samples are taken from the mice to determine if the serum containsantibody to the immunogen. Serum titer is determined by ELISA or RIA.Mice with sera indicating the presence of antibody to the immunogen areselected for hybridoma production.

[0235] Spleens are removed from immune mice and a single cell suspensionis prepared (see Harlow and Lane, 1988). Cell fusions are performedessentially as described by Kohler and Milstein, 1975. Briefly, P3.65.3myeloma cells (American Type Culture Collection, Rockville, Md.) arefused with immune spleen cells using polyethylene glycol as described byHarlow and Lane, 1988. Cells are plated at a density of 2×10⁵ cells/wellin 96 well tissue culture plates. Individual wells are examined forgrowth and the supernatants of wells with growth are tested for thepresence of MMSC2 specific antibodies by ELISA or RIA using wild type ormutant MMSC2 target protein. Cells in positive wells are expanded andsubcloned to establish and confirm monoclonality.

[0236] Clones with the desired specificities are expanded and grown asascites in mice or in a hollow fiber system to produce sufficientquantities of antibody for characterization and assay development.

Example 9 Generation of Monoclonal Antibodies Specific for MMSC2-MMSC2Interacting Protein Complex

[0237] Monoclonal antibodies are generated according to the followingprotocol. Mice are immunized with immunogen comprising MMSC2-MMSC2interacting protein complexes (wild type or mutant), such as MMAC1,conjugated to keyhole limpet hemocyanin using glutaraldehyde or EDC asis well known. The complexes may be stabilized by cross-linking.

[0238] The immunogen is mixed with an adjuvant. Each mouse receives fourinjections of 10 to 100 μg of immunogen and after the fourth injectionblood samples are taken from the mice to determine if the serum containsantibody to the immunogen. Serum titer is determined by ELISA or RIA.Mice with sera indicating the presence of antibody to the immunogen areselected for hybridoma production.

[0239] Spleens are removed from immune mice and a single cell suspensionis prepared (see Harlow and Lane, 1988). Cell fusions are performedessentially as described by Kohler and Milstein, 1975. Briefly, P3.65.3myeloma cells (American Type Culture Collection, Rockville, Md.) arefused with immune spleen cells using polyethylene glycol as described byHarlow and Lane, 1988. Cells are plated at a density of 2×10⁵ cells/wellin 96 well tissue culture plates. Individual wells are examined forgrowth and the supernatants of wells with growth are tested for thepresence of MMSC2-MMSC2 interacting protein complex specific antibodiesby ELISA or RIA using wild type or mutant MMSC2-MMSC2 interactingprotein complexes as target protein. Cells in positive wells areexpanded and subcloned to establish and confirm monoclonality.

[0240] Clones with the desired specificities are expanded and grown asascites in mice or in a hollow fiber system to produce sufficientquantities of antibody for characterization and assay development.Antibodies are tested for binding to MMSC2 alone or to MMSC2 interactingprotein alone to determine which are specific for the complex as opposedto binding to the individual proteins.

Example 10 Sandwich Assay for MMSC2

[0241] Monoclonal antibody is attached to a solid surface such as aplate, tube, bead or particle. Preferably, the antibody is attached tothe well surface of a 96-well ELISA plate. 100 μL sample (e.g., serum,urine, tissue cytosol) containing the MMSC2 peptide/protein (wild-typeor mutants) is added to the solid phase antibody. The sample isincubated for 2 hrs at room temperature. Next the sample fluid isdecanted, and the solid phase is washed with buffer to remove unboundmaterial. 100 μL of a second monoclonal antibody (to a differentdeterminant on the MMSC2 peptide/protein) is added to the solid phase.This antibody is labeled with a detector molecule (e.g., ¹²⁵I, enzyme,fluorophore, or a chromophore) and the solid phase with the secondantibody is incubated for two hrs at room temperature. The secondantibody is decanted and the solid phase is washed with buffer to removeunbound material.

[0242] The amount of bound label, which is proportional to the amount ofMMSC2 peptide/protein present in the sample, is quantified. Separateassays are performed using monoclonal antibodies which are specific forthe wild-type MMSC2 as well as monoclonal antibodies specific for eachof the mutations identified in MMSC2.

Example 11 Sandwich Assay for MMAC1 using MMSC2

[0243] MMSC2 or PDZ domain 7 of MMSC2 is attached to a solid surfacesuch as a plate, tube, bead or particle. Preferably, MMSC2 or its PDZdomain is attached to the well surface of a 96-well ELISA plate. 100 μLsample (e.g., serum, urine, tissue cytosol) containing the MMAC1peptide/protein (wild-type or mutants) is added to the solid phaseMMSC2. The sample is incubated for 2 hrs at room temperature. Next thesample fluid is decanted, and the solid phase is washed with buffer toremove unbound material. 100 μL of a monoclonal antibody to MMAC1 isadded to the solid phase. The antibody is labeled with a detectormolecule (e.g., 1251, enzyme, fluorophore, or a chromophore) and thesolid phase with the antibody is incubated for two hrs at roomtemperature. The antibody is decanted and the solid phase is washed withbuffer to remove unbound material. The amount of bound label, which isproportional to the amount of wild-type MMAC1 present in the sample, isquantified.

Example 12 Drug Screening

[0244] The invention is useful in screening for drugs which can overcomemutations in MMSC2 and also mutations in MMAC1. The knowledge that MMSC2and MMAC1 form a complex is useful in designing such assays. If amutation is present in either MMSC2 or in MMAC1 which prevents theMMSC2-MMAC1 complex from forming, drugs may be screened which willovercome the mutation and allow the protein complex to form and to beactive. Such screening assays can be, e.g., a yeast two hybrid assaywhich is dependent upon two proteins interacting. In such an assay, thepresence of a mutant protein may show no activity or low activity insuch an assay, while the presence of a useful drug will result information of a proper complex which results in activity in the assay.

[0245] A simple binding assay which shows the binding, i.e., formationof a complex, can similarly be used as outlined above. Useful drugs willincrease the formation of MMSC2-MMAC1 complexes. Antibodies may also beused to monitor the amount of complex present. Antibodies specific forthe complex are especially useful. If the presence of a drug increasesthe amount of complex present then the drug is a good candidate fortreating the cancer which is a result of the mutation in either theMMSC2 or the MMAC1.

[0246] While the invention has been disclosed in this patent applicationby reference to the details of preferred embodiments of the invention,it is to be understood that the disclosure is intended in anillustrative rather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

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1 72 1 4 PRT Artificial Sequence Description of ArtificialSequenceConsensus motif for interaction of PDZ domains. 1 Glu Xaa XaaXaa 1 2 7431 DNA Homo sapiens CDS (57)..(6167) 2 gttccatttt aattgctgttaatcatttca gagaagaaca ctgaactttg aaaaaa atg 59 Met 1 ttg gaa gcc att gacaaa aat cgg gcc ctg cat gca gca gag cgc ttg 107 Leu Glu Ala Ile Asp LysAsn Arg Ala Leu His Ala Ala Glu Arg Leu 5 10 15 caa acc aag ctg cga gaacgt ggg gat gta gca aat gaa gac aaa ctg 155 Gln Thr Lys Leu Arg Glu ArgGly Asp Val Ala Asn Glu Asp Lys Leu 20 25 30 agc ctt ctg aag tca gtc ctgcag agc cct ctc ttc agt cag att ctg 203 Ser Leu Leu Lys Ser Val Leu GlnSer Pro Leu Phe Ser Gln Ile Leu 35 40 45 agc ctt cag act tct gta cag cagctg aaa gac cag gta aat att gca 251 Ser Leu Gln Thr Ser Val Gln Gln LeuLys Asp Gln Val Asn Ile Ala 50 55 60 65 act tca gca act tca aat att gaatat gcc cac gtt cct cat ctc agc 299 Thr Ser Ala Thr Ser Asn Ile Glu TyrAla His Val Pro His Leu Ser 70 75 80 cca gct gtg att cct act ctg caa aatgaa tcg ttt tta tta tcc cca 347 Pro Ala Val Ile Pro Thr Leu Gln Asn GluSer Phe Leu Leu Ser Pro 85 90 95 aac aat ggg aat ctg gaa gca ctt aca ggacct ggt att cca cac att 395 Asn Asn Gly Asn Leu Glu Ala Leu Thr Gly ProGly Ile Pro His Ile 100 105 110 aat ggg aaa cct gct tgt gat gaa ttt gatcag ctt atc aaa aat atg 443 Asn Gly Lys Pro Ala Cys Asp Glu Phe Asp GlnLeu Ile Lys Asn Met 115 120 125 gcc cag ggt cgc cat gta gaa gtt ttt gagctc ctc aaa cct cca tct 491 Ala Gln Gly Arg His Val Glu Val Phe Glu LeuLeu Lys Pro Pro Ser 130 135 140 145 gga ggc ctt ggg ttt agt gtt gtg ggacta aga agt gaa aac aga gga 539 Gly Gly Leu Gly Phe Ser Val Val Gly LeuArg Ser Glu Asn Arg Gly 150 155 160 gag ctg gga ata ttt gtt caa gag atacaa gag ggc agt gtg gcc cat 587 Glu Leu Gly Ile Phe Val Gln Glu Ile GlnGlu Gly Ser Val Ala His 165 170 175 aga gat gga aga ttg aaa gaa act gatcaa att ctt gct atc aat gga 635 Arg Asp Gly Arg Leu Lys Glu Thr Asp GlnIle Leu Ala Ile Asn Gly 180 185 190 cag gct ctt gat cag aca att aca catcag cag gct atc agc atc ctg 683 Gln Ala Leu Asp Gln Thr Ile Thr His GlnGln Ala Ile Ser Ile Leu 195 200 205 cag aaa gcc aaa gat act gtc cag ctagtt att gcc aga ggc tca ttg 731 Gln Lys Ala Lys Asp Thr Val Gln Leu ValIle Ala Arg Gly Ser Leu 210 215 220 225 cct cag ctt gtc agc ccc ata gtttcc cgt tct cca tct gca gcc agc 779 Pro Gln Leu Val Ser Pro Ile Val SerArg Ser Pro Ser Ala Ala Ser 230 235 240 aca att tca gct cac tct aat ccggtt cac tgg caa cac atg gaa acg 827 Thr Ile Ser Ala His Ser Asn Pro ValHis Trp Gln His Met Glu Thr 245 250 255 att gaa ttg gtg aat gat gga tctggt ttg gga ttt ggc atc ata gga 875 Ile Glu Leu Val Asn Asp Gly Ser GlyLeu Gly Phe Gly Ile Ile Gly 260 265 270 gga aaa gca act ggt gtg ata gtaaaa acc att ctg cct gga gga gta 923 Gly Lys Ala Thr Gly Val Ile Val LysThr Ile Leu Pro Gly Gly Val 275 280 285 gct gat cag cat ggg cgt tta tgcagt gga gac cac att cta aag att 971 Ala Asp Gln His Gly Arg Leu Cys SerGly Asp His Ile Leu Lys Ile 290 295 300 305 ggt gac aca gat cta gca ggaatg agc agt gag caa gta gca caa gtc 1019 Gly Asp Thr Asp Leu Ala Gly MetSer Ser Glu Gln Val Ala Gln Val 310 315 320 ctt agg caa tgt gga aat agagtt aag ttg atg att gca aga ggt gcc 1067 Leu Arg Gln Cys Gly Asn Arg ValLys Leu Met Ile Ala Arg Gly Ala 325 330 335 ata gaa gaa cgt aca gca cccact gct ttg ggc atc acc ctc tcc tca 1115 Ile Glu Glu Arg Thr Ala Pro ThrAla Leu Gly Ile Thr Leu Ser Ser 340 345 350 tcc cca act tca aca cca gagttg cgg gtt gat gct tct act cag aaa 1163 Ser Pro Thr Ser Thr Pro Glu LeuArg Val Asp Ala Ser Thr Gln Lys 355 360 365 ggt gaa gaa agt gag aca tttgat gta gaa ctc act aaa aat gtc caa 1211 Gly Glu Glu Ser Glu Thr Phe AspVal Glu Leu Thr Lys Asn Val Gln 370 375 380 385 gga tta gga att acc attgct ggc tac att gga gat aaa aaa ttg gaa 1259 Gly Leu Gly Ile Thr Ile AlaGly Tyr Ile Gly Asp Lys Lys Leu Glu 390 395 400 cct tca gga atc ttt gtaaag agc att aca aaa agc agt gcc gtt gag 1307 Pro Ser Gly Ile Phe Val LysSer Ile Thr Lys Ser Ser Ala Val Glu 405 410 415 cat gat gga aga atc caaatt gga gac caa att ata gca gta gat ggc 1355 His Asp Gly Arg Ile Gln IleGly Asp Gln Ile Ile Ala Val Asp Gly 420 425 430 aca aac ctt cag ggt tttact aat cag caa gca gta gag gta ttg cga 1403 Thr Asn Leu Gln Gly Phe ThrAsn Gln Gln Ala Val Glu Val Leu Arg 435 440 445 cat aca gga caa act gtgctc ctg aca cta atg agg aga gga atg aag 1451 His Thr Gly Gln Thr Val LeuLeu Thr Leu Met Arg Arg Gly Met Lys 450 455 460 465 cag gaa gcc gag ctcatg tca agg gaa gac gtc aca aaa gat gca gat 1499 Gln Glu Ala Glu Leu MetSer Arg Glu Asp Val Thr Lys Asp Ala Asp 470 475 480 ttg tct cct gtt aatgcc agc ata atc aaa gaa aat tat gaa aaa gat 1547 Leu Ser Pro Val Asn AlaSer Ile Ile Lys Glu Asn Tyr Glu Lys Asp 485 490 495 gaa gat ttt tta tcttcg acg aga aac acc aac ata tta cca act gaa 1595 Glu Asp Phe Leu Ser SerThr Arg Asn Thr Asn Ile Leu Pro Thr Glu 500 505 510 gaa gaa ggg tat ccatta ctg tca gct gag ata gaa gaa ata gaa gat 1643 Glu Glu Gly Tyr Pro LeuLeu Ser Ala Glu Ile Glu Glu Ile Glu Asp 515 520 525 gca caa aaa caa gaagct gct ctg ctg aca aaa tgg caa agg att atg 1691 Ala Gln Lys Gln Glu AlaAla Leu Leu Thr Lys Trp Gln Arg Ile Met 530 535 540 545 gga att aac tatgaa ata gtg gtg gcc cat gtg agc aag ttt agt gag 1739 Gly Ile Asn Tyr GluIle Val Val Ala His Val Ser Lys Phe Ser Glu 550 555 560 aac agt gga ttgggg ata agc ctg gaa gcg aca gtg gga cat cat ttt 1787 Asn Ser Gly Leu GlyIle Ser Leu Glu Ala Thr Val Gly His His Phe 565 570 575 atc cga tct gttcta cca gag ggt cct gtt gga cac agc ggg aag ctc 1835 Ile Arg Ser Val LeuPro Glu Gly Pro Val Gly His Ser Gly Lys Leu 580 585 590 ttc agt gga gacgag cta ttg gaa gta aat ggc ata act tta ctt ggg 1883 Phe Ser Gly Asp GluLeu Leu Glu Val Asn Gly Ile Thr Leu Leu Gly 595 600 605 gaa aat cac caagat gtg gtg aat atc tta aaa gaa ctg cct ata gaa 1931 Glu Asn His Gln AspVal Val Asn Ile Leu Lys Glu Leu Pro Ile Glu 610 615 620 625 gtg aca atggtg tgc tgt cgt cga act gtg cca ccc acc acc caa tca 1979 Val Thr Met ValCys Cys Arg Arg Thr Val Pro Pro Thr Thr Gln Ser 630 635 640 gaa ttg gatagc ctg gac tta tgt gat att gag cta aca gaa aag cct 2027 Glu Leu Asp SerLeu Asp Leu Cys Asp Ile Glu Leu Thr Glu Lys Pro 645 650 655 cac gta gatcta ggt gag ttc atc ggg tca tca gag aca gag gat cca 2075 His Val Asp LeuGly Glu Phe Ile Gly Ser Ser Glu Thr Glu Asp Pro 660 665 670 gtg ctg gcgatg act gat gcg ggt cag agt aca gaa gag gtt caa gca 2123 Val Leu Ala MetThr Asp Ala Gly Gln Ser Thr Glu Glu Val Gln Ala 675 680 685 cct ttg gccatg tgg gag gct ggc att cag cac ata gag ctg gag aaa 2171 Pro Leu Ala MetTrp Glu Ala Gly Ile Gln His Ile Glu Leu Glu Lys 690 695 700 705 ggg agcaaa gga ctt ggt ttt agc att tta gat tat cag gat cca att 2219 Gly Ser LysGly Leu Gly Phe Ser Ile Leu Asp Tyr Gln Asp Pro Ile 710 715 720 gat ccagca agc act gtg att ata att cgt tct ttg gtg cct ggc ggc 2267 Asp Pro AlaSer Thr Val Ile Ile Ile Arg Ser Leu Val Pro Gly Gly 725 730 735 att gctgaa aag gat gga cga ctt ctt cct ggt gac cga ctc atg ttt 2315 Ile Ala GluLys Asp Gly Arg Leu Leu Pro Gly Asp Arg Leu Met Phe 740 745 750 gta aacgat gtt aac ttg gaa aac agc agt ctt gag gaa gct gta gaa 2363 Val Asn AspVal Asn Leu Glu Asn Ser Ser Leu Glu Glu Ala Val Glu 755 760 765 gca ctgaag gga gca ccg tca ggg act gtg aga ata gga gtt gct aag 2411 Ala Leu LysGly Ala Pro Ser Gly Thr Val Arg Ile Gly Val Ala Lys 770 775 780 785 ccttta ccc ctt tca cca gaa gaa ggt tat gtt tct gct aag gag gat 2459 Pro LeuPro Leu Ser Pro Glu Glu Gly Tyr Val Ser Ala Lys Glu Asp 790 795 800 tccttt ctc tac cca cca cac tcc tgt gag gaa gca ggg ctg gct gac 2507 Ser PheLeu Tyr Pro Pro His Ser Cys Glu Glu Ala Gly Leu Ala Asp 805 810 815 aaaccc ctc ttc agg gct gac ttg gct ctg gtg ggc aca aat gat gct 2555 Lys ProLeu Phe Arg Ala Asp Leu Ala Leu Val Gly Thr Asn Asp Ala 820 825 830 gactta gta gat gaa tcc aca ttt gag tct cca tac tct cct gaa aat 2603 Asp LeuVal Asp Glu Ser Thr Phe Glu Ser Pro Tyr Ser Pro Glu Asn 835 840 845 gacagc atc tac tct act caa gcc tct att tta tct ctt cat ggc agt 2651 Asp SerIle Tyr Ser Thr Gln Ala Ser Ile Leu Ser Leu His Gly Ser 850 855 860 865tct tgt ggt gat ggc ctg aac tat ggt tct tcc ctt cca tca tct cct 2699 SerCys Gly Asp Gly Leu Asn Tyr Gly Ser Ser Leu Pro Ser Ser Pro 870 875 880cct aag gat gtt att gaa aat tct tgt gat cca gta ctt gat ctg cat 2747 ProLys Asp Val Ile Glu Asn Ser Cys Asp Pro Val Leu Asp Leu His 885 890 895atg tct ctg gag gaa cta tat acc cag aat ctc ctg caa aga cag gat 2795 MetSer Leu Glu Glu Leu Tyr Thr Gln Asn Leu Leu Gln Arg Gln Asp 900 905 910gag aat aca cct tcg gtg gac ata agt atg ggg cct gct tct ggc ttt 2843 GluAsn Thr Pro Ser Val Asp Ile Ser Met Gly Pro Ala Ser Gly Phe 915 920 925act ata aat gac tac aca cct gca aat gct att gaa caa caa tat gaa 2891 ThrIle Asn Asp Tyr Thr Pro Ala Asn Ala Ile Glu Gln Gln Tyr Glu 930 935 940945 tgt gaa aac aca ata gtg tgg act gaa tct cat tta cca agt gaa gtt 2939Cys Glu Asn Thr Ile Val Trp Thr Glu Ser His Leu Pro Ser Glu Val 950 955960 ata tca agt gca gaa ctt cct tct gtg cta ccc gat tca gct gga aag 2987Ile Ser Ser Ala Glu Leu Pro Ser Val Leu Pro Asp Ser Ala Gly Lys 965 970975 ggc tct gag tac ctg ctt gaa cag agc tcc ctg gcc tgt aat gct gag 3035Gly Ser Glu Tyr Leu Leu Glu Gln Ser Ser Leu Ala Cys Asn Ala Glu 980 985990 tgt gtc atg ctt caa aat gta tct aaa gaa tct ttt gaa agg act att 3083Cys Val Met Leu Gln Asn Val Ser Lys Glu Ser Phe Glu Arg Thr Ile 995 10001005 aat ata gca aaa ggc aat tct agc cta gga atg aca gtt agt gct aat3131 Asn Ile Ala Lys Gly Asn Ser Ser Leu Gly Met Thr Val Ser Ala Asn1010 1015 1020 1025 aaa gat ggc ttg ggg atg atc gtt cga agc att att catgga ggt gcc 3179 Lys Asp Gly Leu Gly Met Ile Val Arg Ser Ile Ile His GlyGly Ala 1030 1035 1040 att agt cga gat ggc cgg att gcc att ggg gac tgcatc ttg tcc att 3227 Ile Ser Arg Asp Gly Arg Ile Ala Ile Gly Asp Cys IleLeu Ser Ile 1045 1050 1055 aat gaa gag tct acc atc agt gta acc aat gcccag gca cga gct atg 3275 Asn Glu Glu Ser Thr Ile Ser Val Thr Asn Ala GlnAla Arg Ala Met 1060 1065 1070 ttg aga aga cat tct ctc att ggc cct gacata aaa att act tat gtg 3323 Leu Arg Arg His Ser Leu Ile Gly Pro Asp IleLys Ile Thr Tyr Val 1075 1080 1085 cct gca gaa cat ttg gaa gag ttc aaaata agc ttg gga caa caa tct 3371 Pro Ala Glu His Leu Glu Glu Phe Lys IleSer Leu Gly Gln Gln Ser 1090 1095 1100 1105 gga aga gta atg gca ctg gatatt ttt tct tca tac act ggc aga gac 3419 Gly Arg Val Met Ala Leu Asp IlePhe Ser Ser Tyr Thr Gly Arg Asp 1110 1115 1120 att cca gaa tta cca gagcga gaa gag gga gag ggt gaa gaa agc gaa 3467 Ile Pro Glu Leu Pro Glu ArgGlu Glu Gly Glu Gly Glu Glu Ser Glu 1125 1130 1135 ctt caa aac aca gcatat agc aat tgg aat cag ccc agg cgg gtg gaa 3515 Leu Gln Asn Thr Ala TyrSer Asn Trp Asn Gln Pro Arg Arg Val Glu 1140 1145 1150 ctc tgg aga gaacca agc aaa tcc tta ggc atc agc att gtt ggt gga 3563 Leu Trp Arg Glu ProSer Lys Ser Leu Gly Ile Ser Ile Val Gly Gly 1155 1160 1165 cga ggg atgggg agt cgg cta agc aat gga gaa gtg atg agg ggc att 3611 Arg Gly Met GlySer Arg Leu Ser Asn Gly Glu Val Met Arg Gly Ile 1170 1175 1180 1185 ttcatc aaa cat gtt ctg gaa gat agt cca gct ggc aaa aat gga acc 3659 Phe IleLys His Val Leu Glu Asp Ser Pro Ala Gly Lys Asn Gly Thr 1190 1195 1200ttg aaa cct gga gat aga atc gta gag gtg gat gga atg gac ctc aga 3707 LeuLys Pro Gly Asp Arg Ile Val Glu Val Asp Gly Met Asp Leu Arg 1205 12101215 gat gca agc cat gaa caa gct gtg gaa gcc att cgg aaa gca ggc aac3755 Asp Ala Ser His Glu Gln Ala Val Glu Ala Ile Arg Lys Ala Gly Asn1220 1225 1230 cct gta gtc ttt atg gta cag agc att ata aac aga cca agggca ccc 3803 Pro Val Val Phe Met Val Gln Ser Ile Ile Asn Arg Pro Arg AlaPro 1235 1240 1245 agt cag tca gag tca gag cca gag aag gct cca ttg tgcagt gtg ccc 3851 Ser Gln Ser Glu Ser Glu Pro Glu Lys Ala Pro Leu Cys SerVal Pro 1250 1255 1260 1265 cca ccc cct cct tca gcc ttt gcc gaa atg ggtagt gat cac aca cag 3899 Pro Pro Pro Pro Ser Ala Phe Ala Glu Met Gly SerAsp His Thr Gln 1270 1275 1280 tca tct gca agc aaa atc tca caa gat gtggac aaa gag gat gag ttt 3947 Ser Ser Ala Ser Lys Ile Ser Gln Asp Val AspLys Glu Asp Glu Phe 1285 1290 1295 ggt tac agc tgg aaa aat atc aga gagcgt tat gga acc cta aca ggc 3995 Gly Tyr Ser Trp Lys Asn Ile Arg Glu ArgTyr Gly Thr Leu Thr Gly 1300 1305 1310 gag ctg cat atg att gaa ctg gagaaa ggt cat agt ggt ttg ggc cta 4043 Glu Leu His Met Ile Glu Leu Glu LysGly His Ser Gly Leu Gly Leu 1315 1320 1325 agt ctt gct ggg aac aaa gaccga tcc agg atg agt gtc ttc ata gtg 4091 Ser Leu Ala Gly Asn Lys Asp ArgSer Arg Met Ser Val Phe Ile Val 1330 1335 1340 1345 ggg att gat cca aatgga gct gca gga aaa gat ggt cga ttg caa att 4139 Gly Ile Asp Pro Asn GlyAla Ala Gly Lys Asp Gly Arg Leu Gln Ile 1350 1355 1360 gca gat gag cttcta gag atc aat ggt cag att tta tat gga aga agt 4187 Ala Asp Glu Leu LeuGlu Ile Asn Gly Gln Ile Leu Tyr Gly Arg Ser 1365 1370 1375 cat cag aatgcc tca tca atc att aaa tgt gcc cct tct aaa gtg aaa 4235 His Gln Asn AlaSer Ser Ile Ile Lys Cys Ala Pro Ser Lys Val Lys 1380 1385 1390 ata attttt atc aga aat aaa gat gca gtg aat cag atg gcc gta tgt 4283 Ile Ile PheIle Arg Asn Lys Asp Ala Val Asn Gln Met Ala Val Cys 1395 1400 1405 cctgga aat gca gta gaa cct ttg cct tct aac tca gaa aat ctt caa 4331 Pro GlyAsn Ala Val Glu Pro Leu Pro Ser Asn Ser Glu Asn Leu Gln 1410 1415 14201425 aat aag gag aca gag cca act gtt act act tct gat gca gct gtg gac4379 Asn Lys Glu Thr Glu Pro Thr Val Thr Thr Ser Asp Ala Ala Val Asp1430 1435 1440 ctc agt tca ttt aaa aat gtg caa cat ctg gag ctt ccc aaggat cag 4427 Leu Ser Ser Phe Lys Asn Val Gln His Leu Glu Leu Pro Lys AspGln 1445 1450 1455 ggg ggt ttg ggt att gct atc agc gaa gaa gat aca ctcagt gga gtc 4475 Gly Gly Leu Gly Ile Ala Ile Ser Glu Glu Asp Thr Leu SerGly Val 1460 1465 1470 atc ata aag agc tta aca gag cat ggg gta gca gccacg gat gga cga 4523 Ile Ile Lys Ser Leu Thr Glu His Gly Val Ala Ala ThrAsp Gly Arg 1475 1480 1485 ctc aaa gtc gga gat cag ata ctg gct gta gatgat gaa att gtt gtt 4571 Leu Lys Val Gly Asp Gln Ile Leu Ala Val Asp AspGlu Ile Val Val 1490 1495 1500 1505 ggt tac cct att gaa aag ttt att agcctt ctg aag aca gca aag atg 4619 Gly Tyr Pro Ile Glu Lys Phe Ile Ser LeuLeu Lys Thr Ala Lys Met 1510 1515 1520 aca gta aaa ctt acc atc cat gctgag aat cca gat tcc cag gct gtt 4667 Thr Val Lys Leu Thr Ile His Ala GluAsn Pro Asp Ser Gln Ala Val 1525 1530 1535 cct tca gca gct ggt gca gccagt gga gaa aaa aag aac agc tcc cag 4715 Pro Ser Ala Ala Gly Ala Ala SerGly Glu Lys Lys Asn Ser Ser Gln 1540 1545 1550 tct ctg atg gtc cca cagtct ggc tcc cca gaa ccg gag tcc atc cga 4763 Ser Leu Met Val Pro Gln SerGly Ser Pro Glu Pro Glu Ser Ile Arg 1555 1560 1565 aat aca agc aga tcatca aca cca gca att ttt gct tct gat cct gca 4811 Asn Thr Ser Arg Ser SerThr Pro Ala Ile Phe Ala Ser Asp Pro Ala 1570 1575 1580 1585 acc tgc cccatt atc cct ggc tgc gaa aca acc atc gag att tcc aaa 4859 Thr Cys Pro IleIle Pro Gly Cys Glu Thr Thr Ile Glu Ile Ser Lys 1590 1595 1600 ggg cgaaca ggg ctg ggc ctg agc atc gtt ggg ggt tca gac acg ctg 4907 Gly Arg ThrGly Leu Gly Leu Ser Ile Val Gly Gly Ser Asp Thr Leu 1605 1610 1615 ctgggt gcc att att atc cat gaa gtt tat gaa gaa gga gca gca tgt 4955 Leu GlyAla Ile Ile Ile His Glu Val Tyr Glu Glu Gly Ala Ala Cys 1620 1625 1630aaa gat gga aga ctc tgg gct gga gat cag atc tta gag gtg aat gga 5003 LysAsp Gly Arg Leu Trp Ala Gly Asp Gln Ile Leu Glu Val Asn Gly 1635 16401645 att gac ttg aga aag gcc aca cat gat gaa gca atc aat gtc ctg aga5051 Ile Asp Leu Arg Lys Ala Thr His Asp Glu Ala Ile Asn Val Leu Arg1650 1655 1660 1665 cag acg cca cag aga gtg cgc ctg aca ctc tac aga gatgag gcc cca 5099 Gln Thr Pro Gln Arg Val Arg Leu Thr Leu Tyr Arg Asp GluAla Pro 1670 1675 1680 tac aaa gag gag gaa gtg tgt gac acc ctc act attgag ctg cag aag 5147 Tyr Lys Glu Glu Glu Val Cys Asp Thr Leu Thr Ile GluLeu Gln Lys 1685 1690 1695 aag ccg gga aaa ggc cta gga tta agt att gttggt aaa aga aac gat 5195 Lys Pro Gly Lys Gly Leu Gly Leu Ser Ile Val GlyLys Arg Asn Asp 1700 1705 1710 act gga gta ttt gtg tca gac att gtc aaagga gga att gca gat gcc 5243 Thr Gly Val Phe Val Ser Asp Ile Val Lys GlyGly Ile Ala Asp Ala 1715 1720 1725 gat gga aga ctg atg cag gga gac cagata tta atg gtg aat ggg gaa 5291 Asp Gly Arg Leu Met Gln Gly Asp Gln IleLeu Met Val Asn Gly Glu 1730 1735 1740 1745 gac gtt cgt aat gcc acc caagaa gcg gtt gcc gct ttg cta aag tgt 5339 Asp Val Arg Asn Ala Thr Gln GluAla Val Ala Ala Leu Leu Lys Cys 1750 1755 1760 tcc cta ggc aca gta accttg gaa gtt gga aga atc aaa gct ggt cca 5387 Ser Leu Gly Thr Val Thr LeuGlu Val Gly Arg Ile Lys Ala Gly Pro 1765 1770 1775 ttc cat tca gag aggagg cca tct caa agc agc cag gtg agt gaa ggc 5435 Phe His Ser Glu Arg ArgPro Ser Gln Ser Ser Gln Val Ser Glu Gly 1780 1785 1790 agc ctg tca tctttc act ttt cca ctc tct gga tcc agt aca tct gag 5483 Ser Leu Ser Ser PheThr Phe Pro Leu Ser Gly Ser Ser Thr Ser Glu 1795 1800 1805 tca ctg gaaagt agc tca aag aag aat gca ttg gca tct gaa ata cag 5531 Ser Leu Glu SerSer Ser Lys Lys Asn Ala Leu Ala Ser Glu Ile Gln 1810 1815 1820 1825 ggatta aga aca gtc gaa atg aaa aag ggc cct act gac tca ctg gga 5579 Gly LeuArg Thr Val Glu Met Lys Lys Gly Pro Thr Asp Ser Leu Gly 1830 1835 1840atc agc atc gct gga gga gta ggc agc cca ctt ggt gat gtg cct ata 5627 IleSer Ile Ala Gly Gly Val Gly Ser Pro Leu Gly Asp Val Pro Ile 1845 18501855 ttt att gca atg atg cac cca act gga gtt gca gca cag acc caa aaa5675 Phe Ile Ala Met Met His Pro Thr Gly Val Ala Ala Gln Thr Gln Lys1860 1865 1870 ctc aga gtt ggg gat agg att gtc acc atc tgt ggc aca tccact gag 5723 Leu Arg Val Gly Asp Arg Ile Val Thr Ile Cys Gly Thr Ser ThrGlu 1875 1880 1885 ggc atg act cac acc caa gca gtt aac cta ctg aaa aatgca tct ggc 5771 Gly Met Thr His Thr Gln Ala Val Asn Leu Leu Lys Asn AlaSer Gly 1890 1895 1900 1905 tcc att gaa atg cag gtg gtt gct gga gga gacgtg agt gtg gtc aca 5819 Ser Ile Glu Met Gln Val Val Ala Gly Gly Asp ValSer Val Val Thr 1910 1915 1920 ggt cat cag cag gag cct gca agt tcc agtctt tct ttc act ggg ctg 5867 Gly His Gln Gln Glu Pro Ala Ser Ser Ser LeuSer Phe Thr Gly Leu 1925 1930 1935 acg tca agc agt ata ttt cag gat gattta gga cct cct caa tgt aag 5915 Thr Ser Ser Ser Ile Phe Gln Asp Asp LeuGly Pro Pro Gln Cys Lys 1940 1945 1950 tct att aca cta gag cga gga ccagat ggc tta ggc ttc agt ata gtt 5963 Ser Ile Thr Leu Glu Arg Gly Pro AspGly Leu Gly Phe Ser Ile Val 1955 1960 1965 gga gga tat ggc agc cct catgga gac tta ccc att tat gtt aaa aca 6011 Gly Gly Tyr Gly Ser Pro His GlyAsp Leu Pro Ile Tyr Val Lys Thr 1970 1975 1980 1985 gtg ttt gca aag ggagca gcc tct gaa gac gga cgt ctg aaa agg ggc 6059 Val Phe Ala Lys Gly AlaAla Ser Glu Asp Gly Arg Leu Lys Arg Gly 1990 1995 2000 gat cag atc attgct gtc aat ggg cag agt cta gaa gga gtc acc cat 6107 Asp Gln Ile Ile AlaVal Asn Gly Gln Ser Leu Glu Gly Val Thr His 2005 2010 2015 gaa gaa gctgtt gcc atc ctt aaa cgg aca aaa ggc act gtc act ttg 6155 Glu Glu Ala ValAla Ile Leu Lys Arg Thr Lys Gly Thr Val Thr Leu 2020 2025 2030 atg gttctc tct tgaattggct gccagaattg aaccaaccca acccctagct 6207 Met Val Leu Ser2035 cacctcctac tgtaaagaga atgcactggt cctgacaatt tttatgctgt gttcagccgg6267 gtcttcaaaa ctgtaggggg gaaataacac ttaagtttct ttttctcatc tagaaatgct6327 ttccttactg acaacctaac atcatttttc ttttcttctt gcattttgtg aacttaaaga6387 gaaggaatat ttgtgtaggt gaatctcgtt tttatttgtg gagatatcta atgttttgta6447 gtcacatggg caagaattat tacatgctaa gctggttagt ataaagaaag ataattctaa6507 agctaaccaa agaaaatggc ttcagtaaat taggatgaaa aatgaaaata taaaataaag6567 aagaaaatct cggggagttt aaaaaaaatg cctcaatttg gcaatctacc tcctctcccc6627 accccaaact aaaaaaagaa aaaaaggttt tctaatgaaa atctttaaaa atactgtcag6687 tattttaaaa ttttcaacag tattataaaa acattgcatc tccccacctc taatatgcat6747 atatattttt cctgctaaaa ttggtttcta caattgagta aatggcaaat acatgaagca6807 atgtccctaa attttataaa gaaattatat ttaatgcaca tttcaatttt cattcttatt6867 tttgaccttt tataaaatat tttcatgttg ctataagtaa atgatgatgc caccccatgt6927 tgactatggt ttttctagaa agcaactatg ctgctaacca tagaggaaca tagaagggtt6987 ccagaatctt tagtgctggt tttaacaacc gatgcaacat taaaaatgtg ttagtgtgct7047 gtgcaattgg ttttcaattc atattaatct taatgacaga gaacaatgtg ttactaatta7107 ttttggttgt atgccattag taaattgata gaaaaattaa ggggattaac ataacttcat7167 ttcattgcct tatattaaca tcttataata caatagttta agactaaggg aaacagatgg7227 agctgtttat tgagacaact ggtgaggaat tatcatgtgt tcattcccat tttagagcgt7287 gaaactccta cattagaata tataaagtca ctttaaatat ctatatttgt aacagaagta7347 gtgtacagat attttattac agcatttttg tgtaaatgca gaattaaagt gaataaataa7407 gaattttcag tggtgcaaaa aaaa 7431 3 2037 PRT Homo sapiens 3 Met LeuGlu Ala Ile Asp Lys Asn Arg Ala Leu His Ala Ala Glu Arg 1 5 10 15 LeuGln Thr Lys Leu Arg Glu Arg Gly Asp Val Ala Asn Glu Asp Lys 20 25 30 LeuSer Leu Leu Lys Ser Val Leu Gln Ser Pro Leu Phe Ser Gln Ile 35 40 45 LeuSer Leu Gln Thr Ser Val Gln Gln Leu Lys Asp Gln Val Asn Ile 50 55 60 AlaThr Ser Ala Thr Ser Asn Ile Glu Tyr Ala His Val Pro His Leu 65 70 75 80Ser Pro Ala Val Ile Pro Thr Leu Gln Asn Glu Ser Phe Leu Leu Ser 85 90 95Pro Asn Asn Gly Asn Leu Glu Ala Leu Thr Gly Pro Gly Ile Pro His 100 105110 Ile Asn Gly Lys Pro Ala Cys Asp Glu Phe Asp Gln Leu Ile Lys Asn 115120 125 Met Ala Gln Gly Arg His Val Glu Val Phe Glu Leu Leu Lys Pro Pro130 135 140 Ser Gly Gly Leu Gly Phe Ser Val Val Gly Leu Arg Ser Glu AsnArg 145 150 155 160 Gly Glu Leu Gly Ile Phe Val Gln Glu Ile Gln Glu GlySer Val Ala 165 170 175 His Arg Asp Gly Arg Leu Lys Glu Thr Asp Gln IleLeu Ala Ile Asn 180 185 190 Gly Gln Ala Leu Asp Gln Thr Ile Thr His GlnGln Ala Ile Ser Ile 195 200 205 Leu Gln Lys Ala Lys Asp Thr Val Gln LeuVal Ile Ala Arg Gly Ser 210 215 220 Leu Pro Gln Leu Val Ser Pro Ile ValSer Arg Ser Pro Ser Ala Ala 225 230 235 240 Ser Thr Ile Ser Ala His SerAsn Pro Val His Trp Gln His Met Glu 245 250 255 Thr Ile Glu Leu Val AsnAsp Gly Ser Gly Leu Gly Phe Gly Ile Ile 260 265 270 Gly Gly Lys Ala ThrGly Val Ile Val Lys Thr Ile Leu Pro Gly Gly 275 280 285 Val Ala Asp GlnHis Gly Arg Leu Cys Ser Gly Asp His Ile Leu Lys 290 295 300 Ile Gly AspThr Asp Leu Ala Gly Met Ser Ser Glu Gln Val Ala Gln 305 310 315 320 ValLeu Arg Gln Cys Gly Asn Arg Val Lys Leu Met Ile Ala Arg Gly 325 330 335Ala Ile Glu Glu Arg Thr Ala Pro Thr Ala Leu Gly Ile Thr Leu Ser 340 345350 Ser Ser Pro Thr Ser Thr Pro Glu Leu Arg Val Asp Ala Ser Thr Gln 355360 365 Lys Gly Glu Glu Ser Glu Thr Phe Asp Val Glu Leu Thr Lys Asn Val370 375 380 Gln Gly Leu Gly Ile Thr Ile Ala Gly Tyr Ile Gly Asp Lys LysLeu 385 390 395 400 Glu Pro Ser Gly Ile Phe Val Lys Ser Ile Thr Lys SerSer Ala Val 405 410 415 Glu His Asp Gly Arg Ile Gln Ile Gly Asp Gln IleIle Ala Val Asp 420 425 430 Gly Thr Asn Leu Gln Gly Phe Thr Asn Gln GlnAla Val Glu Val Leu 435 440 445 Arg His Thr Gly Gln Thr Val Leu Leu ThrLeu Met Arg Arg Gly Met 450 455 460 Lys Gln Glu Ala Glu Leu Met Ser ArgGlu Asp Val Thr Lys Asp Ala 465 470 475 480 Asp Leu Ser Pro Val Asn AlaSer Ile Ile Lys Glu Asn Tyr Glu Lys 485 490 495 Asp Glu Asp Phe Leu SerSer Thr Arg Asn Thr Asn Ile Leu Pro Thr 500 505 510 Glu Glu Glu Gly TyrPro Leu Leu Ser Ala Glu Ile Glu Glu Ile Glu 515 520 525 Asp Ala Gln LysGln Glu Ala Ala Leu Leu Thr Lys Trp Gln Arg Ile 530 535 540 Met Gly IleAsn Tyr Glu Ile Val Val Ala His Val Ser Lys Phe Ser 545 550 555 560 GluAsn Ser Gly Leu Gly Ile Ser Leu Glu Ala Thr Val Gly His His 565 570 575Phe Ile Arg Ser Val Leu Pro Glu Gly Pro Val Gly His Ser Gly Lys 580 585590 Leu Phe Ser Gly Asp Glu Leu Leu Glu Val Asn Gly Ile Thr Leu Leu 595600 605 Gly Glu Asn His Gln Asp Val Val Asn Ile Leu Lys Glu Leu Pro Ile610 615 620 Glu Val Thr Met Val Cys Cys Arg Arg Thr Val Pro Pro Thr ThrGln 625 630 635 640 Ser Glu Leu Asp Ser Leu Asp Leu Cys Asp Ile Glu LeuThr Glu Lys 645 650 655 Pro His Val Asp Leu Gly Glu Phe Ile Gly Ser SerGlu Thr Glu Asp 660 665 670 Pro Val Leu Ala Met Thr Asp Ala Gly Gln SerThr Glu Glu Val Gln 675 680 685 Ala Pro Leu Ala Met Trp Glu Ala Gly IleGln His Ile Glu Leu Glu 690 695 700 Lys Gly Ser Lys Gly Leu Gly Phe SerIle Leu Asp Tyr Gln Asp Pro 705 710 715 720 Ile Asp Pro Ala Ser Thr ValIle Ile Ile Arg Ser Leu Val Pro Gly 725 730 735 Gly Ile Ala Glu Lys AspGly Arg Leu Leu Pro Gly Asp Arg Leu Met 740 745 750 Phe Val Asn Asp ValAsn Leu Glu Asn Ser Ser Leu Glu Glu Ala Val 755 760 765 Glu Ala Leu LysGly Ala Pro Ser Gly Thr Val Arg Ile Gly Val Ala 770 775 780 Lys Pro LeuPro Leu Ser Pro Glu Glu Gly Tyr Val Ser Ala Lys Glu 785 790 795 800 AspSer Phe Leu Tyr Pro Pro His Ser Cys Glu Glu Ala Gly Leu Ala 805 810 815Asp Lys Pro Leu Phe Arg Ala Asp Leu Ala Leu Val Gly Thr Asn Asp 820 825830 Ala Asp Leu Val Asp Glu Ser Thr Phe Glu Ser Pro Tyr Ser Pro Glu 835840 845 Asn Asp Ser Ile Tyr Ser Thr Gln Ala Ser Ile Leu Ser Leu His Gly850 855 860 Ser Ser Cys Gly Asp Gly Leu Asn Tyr Gly Ser Ser Leu Pro SerSer 865 870 875 880 Pro Pro Lys Asp Val Ile Glu Asn Ser Cys Asp Pro ValLeu Asp Leu 885 890 895 His Met Ser Leu Glu Glu Leu Tyr Thr Gln Asn LeuLeu Gln Arg Gln 900 905 910 Asp Glu Asn Thr Pro Ser Val Asp Ile Ser MetGly Pro Ala Ser Gly 915 920 925 Phe Thr Ile Asn Asp Tyr Thr Pro Ala AsnAla Ile Glu Gln Gln Tyr 930 935 940 Glu Cys Glu Asn Thr Ile Val Trp ThrGlu Ser His Leu Pro Ser Glu 945 950 955 960 Val Ile Ser Ser Ala Glu LeuPro Ser Val Leu Pro Asp Ser Ala Gly 965 970 975 Lys Gly Ser Glu Tyr LeuLeu Glu Gln Ser Ser Leu Ala Cys Asn Ala 980 985 990 Glu Cys Val Met LeuGln Asn Val Ser Lys Glu Ser Phe Glu Arg Thr 995 1000 1005 Ile Asn IleAla Lys Gly Asn Ser Ser Leu Gly Met Thr Val Ser Ala 1010 1015 1020 AsnLys Asp Gly Leu Gly Met Ile Val Arg Ser Ile Ile His Gly Gly 1025 10301035 1040 Ala Ile Ser Arg Asp Gly Arg Ile Ala Ile Gly Asp Cys Ile LeuSer 1045 1050 1055 Ile Asn Glu Glu Ser Thr Ile Ser Val Thr Asn Ala GlnAla Arg Ala 1060 1065 1070 Met Leu Arg Arg His Ser Leu Ile Gly Pro AspIle Lys Ile Thr Tyr 1075 1080 1085 Val Pro Ala Glu His Leu Glu Glu PheLys Ile Ser Leu Gly Gln Gln 1090 1095 1100 Ser Gly Arg Val Met Ala LeuAsp Ile Phe Ser Ser Tyr Thr Gly Arg 1105 1110 1115 1120 Asp Ile Pro GluLeu Pro Glu Arg Glu Glu Gly Glu Gly Glu Glu Ser 1125 1130 1135 Glu LeuGln Asn Thr Ala Tyr Ser Asn Trp Asn Gln Pro Arg Arg Val 1140 1145 1150Glu Leu Trp Arg Glu Pro Ser Lys Ser Leu Gly Ile Ser Ile Val Gly 11551160 1165 Gly Arg Gly Met Gly Ser Arg Leu Ser Asn Gly Glu Val Met ArgGly 1170 1175 1180 Ile Phe Ile Lys His Val Leu Glu Asp Ser Pro Ala GlyLys Asn Gly 1185 1190 1195 1200 Thr Leu Lys Pro Gly Asp Arg Ile Val GluVal Asp Gly Met Asp Leu 1205 1210 1215 Arg Asp Ala Ser His Glu Gln AlaVal Glu Ala Ile Arg Lys Ala Gly 1220 1225 1230 Asn Pro Val Val Phe MetVal Gln Ser Ile Ile Asn Arg Pro Arg Ala 1235 1240 1245 Pro Ser Gln SerGlu Ser Glu Pro Glu Lys Ala Pro Leu Cys Ser Val 1250 1255 1260 Pro ProPro Pro Pro Ser Ala Phe Ala Glu Met Gly Ser Asp His Thr 1265 1270 12751280 Gln Ser Ser Ala Ser Lys Ile Ser Gln Asp Val Asp Lys Glu Asp Glu1285 1290 1295 Phe Gly Tyr Ser Trp Lys Asn Ile Arg Glu Arg Tyr Gly ThrLeu Thr 1300 1305 1310 Gly Glu Leu His Met Ile Glu Leu Glu Lys Gly HisSer Gly Leu Gly 1315 1320 1325 Leu Ser Leu Ala Gly Asn Lys Asp Arg SerArg Met Ser Val Phe Ile 1330 1335 1340 Val Gly Ile Asp Pro Asn Gly AlaAla Gly Lys Asp Gly Arg Leu Gln 1345 1350 1355 1360 Ile Ala Asp Glu LeuLeu Glu Ile Asn Gly Gln Ile Leu Tyr Gly Arg 1365 1370 1375 Ser His GlnAsn Ala Ser Ser Ile Ile Lys Cys Ala Pro Ser Lys Val 1380 1385 1390 LysIle Ile Phe Ile Arg Asn Lys Asp Ala Val Asn Gln Met Ala Val 1395 14001405 Cys Pro Gly Asn Ala Val Glu Pro Leu Pro Ser Asn Ser Glu Asn Leu1410 1415 1420 Gln Asn Lys Glu Thr Glu Pro Thr Val Thr Thr Ser Asp AlaAla Val 1425 1430 1435 1440 Asp Leu Ser Ser Phe Lys Asn Val Gln His LeuGlu Leu Pro Lys Asp 1445 1450 1455 Gln Gly Gly Leu Gly Ile Ala Ile SerGlu Glu Asp Thr Leu Ser Gly 1460 1465 1470 Val Ile Ile Lys Ser Leu ThrGlu His Gly Val Ala Ala Thr Asp Gly 1475 1480 1485 Arg Leu Lys Val GlyAsp Gln Ile Leu Ala Val Asp Asp Glu Ile Val 1490 1495 1500 Val Gly TyrPro Ile Glu Lys Phe Ile Ser Leu Leu Lys Thr Ala Lys 1505 1510 1515 1520Met Thr Val Lys Leu Thr Ile His Ala Glu Asn Pro Asp Ser Gln Ala 15251530 1535 Val Pro Ser Ala Ala Gly Ala Ala Ser Gly Glu Lys Lys Asn SerSer 1540 1545 1550 Gln Ser Leu Met Val Pro Gln Ser Gly Ser Pro Glu ProGlu Ser Ile 1555 1560 1565 Arg Asn Thr Ser Arg Ser Ser Thr Pro Ala IlePhe Ala Ser Asp Pro 1570 1575 1580 Ala Thr Cys Pro Ile Ile Pro Gly CysGlu Thr Thr Ile Glu Ile Ser 1585 1590 1595 1600 Lys Gly Arg Thr Gly LeuGly Leu Ser Ile Val Gly Gly Ser Asp Thr 1605 1610 1615 Leu Leu Gly AlaIle Ile Ile His Glu Val Tyr Glu Glu Gly Ala Ala 1620 1625 1630 Cys LysAsp Gly Arg Leu Trp Ala Gly Asp Gln Ile Leu Glu Val Asn 1635 1640 1645Gly Ile Asp Leu Arg Lys Ala Thr His Asp Glu Ala Ile Asn Val Leu 16501655 1660 Arg Gln Thr Pro Gln Arg Val Arg Leu Thr Leu Tyr Arg Asp GluAla 1665 1670 1675 1680 Pro Tyr Lys Glu Glu Glu Val Cys Asp Thr Leu ThrIle Glu Leu Gln 1685 1690 1695 Lys Lys Pro Gly Lys Gly Leu Gly Leu SerIle Val Gly Lys Arg Asn 1700 1705 1710 Asp Thr Gly Val Phe Val Ser AspIle Val Lys Gly Gly Ile Ala Asp 1715 1720 1725 Ala Asp Gly Arg Leu MetGln Gly Asp Gln Ile Leu Met Val Asn Gly 1730 1735 1740 Glu Asp Val ArgAsn Ala Thr Gln Glu Ala Val Ala Ala Leu Leu Lys 1745 1750 1755 1760 CysSer Leu Gly Thr Val Thr Leu Glu Val Gly Arg Ile Lys Ala Gly 1765 17701775 Pro Phe His Ser Glu Arg Arg Pro Ser Gln Ser Ser Gln Val Ser Glu1780 1785 1790 Gly Ser Leu Ser Ser Phe Thr Phe Pro Leu Ser Gly Ser SerThr Ser 1795 1800 1805 Glu Ser Leu Glu Ser Ser Ser Lys Lys Asn Ala LeuAla Ser Glu Ile 1810 1815 1820 Gln Gly Leu Arg Thr Val Glu Met Lys LysGly Pro Thr Asp Ser Leu 1825 1830 1835 1840 Gly Ile Ser Ile Ala Gly GlyVal Gly Ser Pro Leu Gly Asp Val Pro 1845 1850 1855 Ile Phe Ile Ala MetMet His Pro Thr Gly Val Ala Ala Gln Thr Gln 1860 1865 1870 Lys Leu ArgVal Gly Asp Arg Ile Val Thr Ile Cys Gly Thr Ser Thr 1875 1880 1885 GluGly Met Thr His Thr Gln Ala Val Asn Leu Leu Lys Asn Ala Ser 1890 18951900 Gly Ser Ile Glu Met Gln Val Val Ala Gly Gly Asp Val Ser Val Val1905 1910 1915 1920 Thr Gly His Gln Gln Glu Pro Ala Ser Ser Ser Leu SerPhe Thr Gly 1925 1930 1935 Leu Thr Ser Ser Ser Ile Phe Gln Asp Asp LeuGly Pro Pro Gln Cys 1940 1945 1950 Lys Ser Ile Thr Leu Glu Arg Gly ProAsp Gly Leu Gly Phe Ser Ile 1955 1960 1965 Val Gly Gly Tyr Gly Ser ProHis Gly Asp Leu Pro Ile Tyr Val Lys 1970 1975 1980 Thr Val Phe Ala LysGly Ala Ala Ser Glu Asp Gly Arg Leu Lys Arg 1985 1990 1995 2000 Gly AspGln Ile Ile Ala Val Asn Gly Gln Ser Leu Glu Gly Val Thr 2005 2010 2015His Glu Glu Ala Val Ala Ile Leu Lys Arg Thr Lys Gly Thr Val Thr 20202025 2030 Leu Met Val Leu Ser 2035 4 15 PRT Homo sapiens 4 Asn Glu ProPhe Asp Glu Asp Gln His Thr Gln Ile Thr Lys Val 1 5 10 15 5 21 DNA Homosapiens 5 agacagcaaa gatgacagta a 21 6 20 DNA Homo sapiens 6 cttcctcctctttgtatggg 20 7 21 DNA Homo sapiens 7 gcttttgccg aaatgggtag t 21 8 21DNA Homo sapiens 8 gatcggtctt tgttcccagc a 21 9 19 DNA Homo sapiens 9tgtgagcaag tttagtgag 19 10 19 DNA Homo sapiens 10 ggtgattttc cccaagtaa19 11 16 PRT Homo sapiens 11 Ser Gly Ser Gly Ile Leu Ala Pro Pro Val ProPro Arg Asn Thr Arg 1 5 10 15 12 16 PRT Homo sapiens 12 Glu Asn Glu ProPhe Asp Glu Asp Gln His Thr Gln Ile Thr Lys Val 1 5 10 15 13 22 DNA Homosapiens 13 gccaccgcgg gattaagttt ct 22 14 23 DNA Homo sapiens 14tgtagccagc aatggtaatt cct 23 15 41 DNA Homo sapiens 15 gttttcccagtcacgacggt tccattttaa ttgctgttaa t 41 16 43 DNA Homo sapiens 16aggaaacagc tatgaccatg gggataataa aaacgattca ggg 43 17 39 DNA Homosapiens 17 gttttcccag tcacgacgtt gaatatgccc acgttcctc 39 18 42 DNA Homosapiens 18 aggaaacagc tatgaccatt ctttcaatct tccatctcta tg 42 19 39 DNAHomo sapiens 19 gttttcccag tcacgacgtt gaatatgccc acgttcctc 39 20 41 DNAHomo sapiens 20 aggaaacagc tatgaccatc aaaccagatc catcattcac c 41 21 40DNA Homo sapiens 21 gttttcccag tcacgacggc acaatttcag ctcactctaa 40 22 39DNA Homo sapiens 22 aggaaacagc tatgaccatg gatgaggaga gggtgatgc 39 23 22DNA Homo sapiens 23 tctagcagga atgagcagtg ag 22 24 24 DNA Homo sapiens24 gatcctgata atctaaaatg ctaa 24 25 40 DNA Homo sapiens 25 gttttcccagtcacgacgaa gttgatgatt gcaagaggtg 40 26 41 DNA Homo sapiens 26 aggaaacagctatgaccatg gtttgtgcca tctactgcta t 41 27 39 DNA Homo sapiens 27gttttcccag tcacgacgaa acgagtgccg ttgagcatg 39 28 40 DNA Homo sapiens 28aggaaacagc tatgaccatg ctgacagtaa tggataccct 40 29 41 DNA Homo sapiens 29gttttcccag tcacgacgga ttttttatct tcgacgagaa a 41 30 42 DNA Homo sapiens30 aggaaacagc tatgaccatt tccccaagta aagttatgcc at 42 31 38 DNA Homosapiens 31 gttttcccag tcacgacgtc ctgttggaca cagcggga 38 32 39 DNA Homosapiens 32 aggaaacagc tatgaccatc atggccaaag gtgcttgaa 39 33 22 DNA Homosapiens 33 ccacccacca cccaatcaga at 22 34 22 DNA Homo sapiens 34catctcgact aatggcacct cc 22 35 38 DNA Homo sapiens 35 gttttcccagtcacgacgga gacagaggat ccagtgct 38 36 39 DNA Homo sapiens 36 aggaaacagctatgaccatc cctgacggtg ctcccttca 39 37 40 DNA Homo sapiens 37 gttttcccagtcacgacgtt aacttggaaa acagcagtct 40 38 41 DNA Homo sapiens 38 aggaaacagctatgaccatc atcaccacaa gaactgccat g 41 39 40 DNA Homo sapiens 39gttttcccag tcacgacgac tctcctgaaa atgacagcat 40 40 41 DNA Homo sapiens 40aggaaacagc tatgaccatt aaatgagatt cagtccacac t 41 41 40 DNA Homo sapiens41 gttttcccag tcacgacgat aaatgactac acacctgcaa 40 42 41 DNA Homo sapiens42 aggaaacagc tatgaccata acgatcatcc ccaagccatc t 41 43 22 DNA Homosapiens 43 ctgagtacct gcttgaacag ag 22 44 22 DNA Homo sapiens 44gaccattgat ctctagaagc tc 22 45 41 DNA Homo sapiens 45 gttttcccagtcacgacggg actattaata tagcaaaagg c 41 46 40 DNA Homo sapiens 46aggaaacagc tatgaccatc agtgccatta ctcttccaga 40 47 39 DNA Homo sapiens 47gttttcccag tcacgacgta cttatgtgcc tgcagaaca 39 48 40 DNA Homo sapiens 48aggaaacagc tatgaccatc atgtttgatg aaaatgcccc 40 49 39 DNA Homo sapiens 49gttttcccag tcacgacgat tgttggtgga cgagggatg 39 50 40 DNA Homo sapiens 50aggaaacagc tatgaccatc catttcggca aaggctgaag 40 51 39 DNA Homo sapiens 51gttttcccag tcacgacgca gagtcagagc cagagaagg 39 52 40 DNA Homo sapiens 52aggaaacagc tatgaccata gaagctcagc tgcaatttgc 40 53 21 DNA Homo sapiens 53caggcgagct gcatatgatt g 21 54 23 DNA Homo sapiens 54 cctcctttgacaatgtctga cac 23 55 41 DNA Homo sapiens 55 gttttcccag tcacgacggtgtcttcatag tggggattga t 41 56 40 DNA Homo sapiens 56 aggaaacagctatgaccatg aagctccaga tgttgcacat 40 57 39 DNA Homo sapiens 57 gttttcccagtcacgacgag agccaactgt tactacttc 39 58 40 DNA Homo sapiens 58 aggaaacagctatgaccatt gaaggaacag cctgggaatc 40 59 39 DNA Homo sapiens 59 gttttcccagtcacgacgtt agccttctga agacagcaa 39 60 40 DNA Homo sapiens 60 aggaaacagctatgaccatc atggataata atggcaccca 40 61 39 DNA Homo sapiens 61 gttttcccagtcacgacgtt tccaaagggc gaacagggc 39 62 41 DNA Homo sapiens 62 aggaaacagctatgaccatc caacaatact taatcctagg c 41 63 23 DNA Homo sapiens 63tggaattgac ttgagaaagg cca 23 64 22 DNA Homo sapiens 64 ccccctacagttttgaagac cc 22 65 40 DNA Homo sapiens 65 gttttcccag tcacgacgaagaggaggaag tgtgtgacac 40 66 40 DNA Homo sapiens 66 aggaaacagc tatgaccatgacaggctgcc ttcactcacc 40 67 39 DNA Homo sapiens 67 gttttcccag tcacgacgtcaaagctggtc cattccatt 39 68 40 DNA Homo sapiens 68 aggaaacagc tatgaccatggatgtgccac agatggtgac 40 69 39 DNA Homo sapiens 69 gttttcccag tcacgacgatgatgcaccca actggagtt 39 70 40 DNA Homo sapiens 70 aggaaacagc tatgaccatggctgccatat cctccaacta 40 71 39 DNA Homo sapiens 71 gttttcccag tcacgacgggacctcctcaa tgtaagtct 39 72 40 DNA Homo sapiens 72 aggaaacagc tatgaccatattgtcaggac cagtgcattc 40

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: (a) a human MMSC2 polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 3; (b) a mutated human MMSC2polypeptide obtainable by expression of a mutated form of the nucleicacid set forth in SEQ ID NO: 2; and (c) a mutant human MMSC2 polypeptidewhich cannot form a complex with a wild-type protein with whichwild-type MMSC2 does form a complex.
 2. The isolated polypeptide ofclaim 1 which is a human MMSC2 polypeptide comprising the amino acidsequence set forth in SEQ ID NO:
 3. 3. The isolated polypeptide of claim1 which is a mutated human MMSC2 polypeptide obtainable by expression ofa mutated form of the nucleic acid set forth in SEQ ID NO:
 2. 4. Theisolated polypeptide of claim 3, wherein said mutated human MMSC2polypeptide comprises a mutation selected from the group consisting of:an Ile at amino acid 55; a Trp at amino acid 115; a Thr at amino acid358; and a Lys at amino acid 1875 with respect to the amino acidsequence set forth in SEQ ID NO:
 3. 5. The isolated polypeptide of claim1 which is an isolated mutant human MMSC2 polypeptide which cannot forma complex with a wild-type protein with which wild-type MMSC2 does forma complex.
 6. The isolated polypeptide of claim 4, wherein saidwild-type protein is MMAC1.
 7. The isolated polypeptide of claim 1 whichis labeled.
 8. The isolated polypeptide of claim 1 in the form of afusion protein.
 9. An isolated protein complex selected from the groupconsisting of: (a) a protein complex comprising MMSC2 and MMAC1 and (b)a protein complex comprising a fragment of MMSC2 and a fragment ofMMAC1.
 10. The isolated protein complex of claim 9 which is proteincomplex comprising MMSC2 and MMAC1.
 11. The isolated protein complex ofclaim 10, wherein said MMSC2 contains an alteration.
 12. The isolatedprotein complex of claim 10, wherein said MMAC1 contains an alteration.13. The isolated protein complex of claim 9 which is a complex of afragment of MMSC2 and a fragment of MMAC1.
 14. The protein complex ofclaim 13, wherein said fragment of MMSC2 comprises PDZ domain number 7.15. The protein complex of claim 13, wherein said MMSC2 comprises analteration.
 16. The protein complex of claim 13, wherein said MMAC1comprises an alteration.
 17. The protein complex of claim 14, whereinsaid MMSC2 comprises an alteration.
 18. The protein complex of claim 14,wherein said MMAC1 comprises an alteration.
 19. A method for detectingan alteration in MMSC2 wherein said alteration is associated with cancerin a human, wherein if said alteration is in germline it is associatedwith predisposition to said cancer and if said alteration is in somatictissue it indicates that said somatic tissue is cancerous, wherein saidmethod comprises analyzing a MMSC2 gene expression product from a tissueof said human.
 20. The method of claim 19, wherein said expressionproduct is selected from the group consisting of a MMSC2 polypeptideencoded by the MMSC2 gene.
 21. The method of claim 20 wherein one ormore of the following procedures is carried out: (a) immunoblotting; (b)immunocytochemistry; (c) assaying for binding interactions between MMSC2protein isolated from said tissue and a binding partner capable ofspecifically binding the polypeptide expression product of a MMSC2mutant allele and/or a binding partner for the MMSC2 polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 3; and (d) assaying forthe inhibition of biochemical activity of said binding partner.
 22. Themethod of claim 21 wherein said alteration of MMSC2 protein is detectedby assaying for binding interactions between said MMSC2 protein isolatedfrom said tissue and MMAC1 protein.
 23. A method for detecting analteration in MMAC1 wherein said alteration is associated with cancer ina human, wherein if said alteration is in germline it is associated withpredisposition to said cancer and if said alteration is in somatictissue it indicates that said somatic tissue is cancerous, wherein saidmethod comprises analyzing an MMAC1 polypeptide from a tissue of saidhuman by assaying for binding interactions between said MMAC1polypeptide and MMSC2 or PDZ domain number 7 of said MMSC2.
 24. A methodfor supplying a wild-type MMSC2 gene function or a MMSC2 functionsubstantially similar to wild-type to a cell which has lost said genefunction or has altered gene function by virtue of a mutation in saidMMSC2 gene, wherein said method comprises introducing into said cell amolecule which suppresses a transformed state of said cell, saidmolecule selected from the group consisting of all or a part of awild-type MMSC2 polypeptide which is required for non-neoplastic growthof said cell, a polypeptide substanially homologous to said wild-typeMMSC2 polypeptide and a molecule which mimics the function of saidwild-type MMSC2 polypeptide.
 25. A method for diagnosing apredisposition for cancer in a human wherein said method comprisesassaying for the ability of MMSC2 or a fragment of MMSC2 from said humanto form a complex with a protein to which wild-type MMSC2 binds whereinan inability to form said complex is indicative of a predisposition tocancer.
 26. The method of claim 25, wherein said protein is MMAC1. 27.The method of claim 25, wherein said assay comprises measuring in vitroa complex formed by mixing said protein and MMSC2 purified from saidhuman.
 28. The method of claim 25, wherein said assay comprisesmeasuring in vitro a complex formed by mixing MMSC2 and said proteinpurified from said human.
 29. The method of claim 25, wherein saidcomplex is measured by binding with an antibody specific for aMMSC2-said protein complex.
 30. The method of claim 25, wherein saidassay comprises mixing an antibody specific for a MMSC2-said proteincomplex with a tissue extract from said person, wherein the lack offormation of a MMSC2-said protein-antibody complex between said antibodyand said tissue extract is indicative of a predisposition to cancer. 31.A method for determining whether a mutation in a protein to which MMSC2binds is predispositive for cancer wherein said method comprises bindingsaid protein with said mutation to a wild-type MMSC2 and determiningwhether a complex forms, wherein the lack of a complex indicates saidmutation is predispositive.
 32. A method for determining whether amutation in MMSC2 is predispositive for cancer wherein said methodcomprises binding a MMSC2 with said mutation to a protein to whichwild-type MMSC2 binds and determining whether a complex forms, whereinthe lack of a complex indicates said mutation is predispositive.
 33. Amethod for treating a human with cancer resulting from a mutation in aprotein to which MMSC2 binds and wherein said mutation prevents bindingof said protein to MMSC2 wherein said method comprises treating saidperson with a complex of wild-type MMSC2 and wild-type of said protein.34. The method of claim 33 wherein said protein is MMAC1.